3,5-disubstituted and 3,5,7-trisubstituted-3h-oxazolo and 3h-thiazolo[4,5-d]pyrimidin-2-one compounds and prodrugs thereof

ABSTRACT

The invention is directed to 3,5-disubstituted and 3,5,7-trisubstituted-3H-oxazolo and 3H-thiazolo[4,5-d]pyrimidin-2-one compounds and prodrugs thereof that have immunomodulatory activity. The invention is also directed to the therapeutic or prophylactic use of such compounds and pharmaceutical compositions containing them, and to methods of treating diseases and disorders described herein, by administering effective amounts of such compounds and prodrugs.

This application is a divisional application of and claims priority toU.S. patent application Ser. No. 11/304,691 filed Dec. 16, 2005, whichclaims the benefit of U.S. Provisional Application No. 60/636,633, filedDec. 17, 2004, and U.S. Provisional Application No. 60/636,634, filedDec. 17, 2004.

FIELD OF THE INVENTION

The invention is directed to 3,5-disubstituted and3,5,7-trisubstituted-3H-oxazolo and 3H-thiazolo[4,5-d]pyrimidin-2-onecompounds and prodrugs thereof that have immunomodulatory activity. Theinvention is also directed to the therapeutic or prophylactic use ofsuch compounds and pharmaceutical compositions containing them, and tomethods of treating diseases and disorders described herein, byadministering effective amounts of such compounds and prodrugs.

BACKGROUND OF THE INVENTION

The last few decades have seen significant efforts expended in exploringpossible therapeutic uses of guanine analogs and nucleosides thereof. Anumber of nucleoside analogs are currently being marketed as antiviraldrugs, including HIV reverse transcriptase inhibitors such as AZT, ddI,ddC, d4T, 3TC and the guanosine nucleoside analog abacavir. While notadhering to a particular theory, nucleoside analogs may provide benefitsby directly inhibiting the pathogen or tumor, by stimulation of hostimmune functions, or some combination of these or other mechanisms.

One of the studied guanosine analogs with demonstrated immunomodulatoryactivity is5-amino-3-(β-D-ribofuranosylthiazolo[4,5-d]pyrimidine-2,7(3H,6H)dione(7-thia-8-oxoguanosine).For example, certain pyrimido[4,5-d]pyrimidine nucleosides are disclosedin U.S. Pat. No. 5,041,542 to Robins et al. as being effective intreatment against L1210 in BDF1 mice. In addition,3-β-D-ribofuranosylthiazolo[4,5-d]pyrimidines demonstrating significantimmunoactivity, including murine spleen cell proliferation and in vivoactivity against Semliki Forest virus, are disclosed U.S. Pat. Nos.5,041,426 and 4,880,784 to Robins et al. A number of publications havealso described non-glycosyl derivatives of the thiazolo[4,5-d]pyrimidinemoiety. See, e.g., U.S. Pat. Nos. 5,994,321 and 5,446,045; Revankar etal., J. Het. Chem., 30, 1341-49 (1993); Lewis et al., J. Het. Chem., 32,547-56 (1995).

SUMMARY OF THE INVENTION

The present invention describes novel 3,5-disubstituted and3,5,7-trisubstituted-3H-oxazolo and 3H-thiazolo[4,5-d]pyrimidin-2-onecompounds, pharmaceutically active prodrugs, pharmaceutically activemetabolites, pharmaceutically acceptable salts, and pharmaceuticallyacceptable solvates thereof, which are useful as immunomodulators.

In another embodiment, the present invention encompasses a method oftreating or preventing a hepatitis C virus infection in a patient inneed thereof comprising administering to the patient a therapeuticallyor prophylactically effective amount of a 3,5-disubstituted and3,5,7-trisubstituted-3H-oxazolo and 3H-thiazolo[4,5-d]pyrimidin-2-onecompound or a prodrug thereof.

In a general aspect, the invention relates to prodrugs that are3,5-disubstituted and 3,5,7-trisubstituted-3H-oxazolo and3H-thiazolo[4,5-d]pyrimidin-2-one compounds of Formula I

wherein

X is O or S, Y is O or S,

R¹ is H, alkyl, aryl, cycloalkyl, or heterocyclyl,

R² is NH₂, —NHC(O)R⁴, —NHR⁵, —N═CHNR⁶R⁷, R³ is H, Cl, Br, or OR⁸,

R⁴ is —C₁-C₇-alkyl or —O(C₁-C₇-alkyl),R⁵ is —C₁-C₇-alkyl,R⁶ and R⁷ are independently —C₁-C₇-alkyl or together with nitrogen forma 5- or 6-membered heterocyclic ring,

R⁸ is —CHR⁹R¹⁰,

R⁹ is H, —C₁-C₇-alkyl, cycloalkyl, aryl, heterocyclyl, —NR¹¹R¹², or OR⁵,R¹⁰ is —C₁-C₇-alkyl, cycloalkyl, aryl, heterocyclyl, —NR¹¹R¹², or OR⁵,R¹¹ and R¹² are independently H, —C₁-C₇-alkyl, or —C(O)R⁴,wherein when X is O, Y is S, and R³ is H, Cl, Br, or OR⁸, R¹ is not H orβ-D-ribose or esters thereof,wherein the above alkyl, aryl, cycloalkyl, or heterocyclyl moieties areoptionally substituted by 1-4 substituents selected from

-   -   hydrogen,    -   alkanoyl,    -   alkylamine,    -   amino,    -   aryl, cycloalkyl, heterocyclyl,    -   azido,    -   C₁-C₆ alkyl, C₁-C₆ haloalkyl, C₁-C₆ hydroxyalkyl, C₁-C₆ alkoxy,        C₁-C₆ alkylamine, C₁-C₆ dialkylamine, C₂-C₆ alkenyl, or C₂-C₆        alkynyl, wherein each of which may be interrupted by one or more        hetero atoms,    -   carboxyl,    -   cyano,    -   halo,    -   hydroxy,    -   mercapto,    -   nitro,    -   thioalkyl,    -   —N═N—NH₂,    -   —C(O)₂—(C₁-C₆ alkyl), —C(O)₂-(aryl), —C(O)₂-(cycloalkyl),        —C(O)₂-(heterocyclyl), —O—(C₁-C₆ haloalkyl), —O—(C₁-C₆        alkyl)aryl, —O—(C₁-C₆ alkyl)cycloalkyl, —O—(C₁-C₆        alkyl)heterocyclyl, —O—(C₁-C₆ alkyl)amino, —O—(C₁-C₆        alkyl)alkylamino, —O—(C₁-C₆ alkyl)dialkylamino, —O—(C₁-C₆        alkyl)-C(O)-amino, —O—(C₁-C₆ alkyl)-C(O)-alkylamino, —O—(C₁-C₆        alkyl)-S(O)₂-amino, —O—(C₁-C₆ alkyl)-S(O)₂-alkylamino, —O—(C₁-C₆        alkyl)-S(O)₂-dialkylamino, —O—(C₁-C₆ alkyl)-C(O)-dialkylamino,        —O-aryl, —O-heterocyclyl, —NHC(O)—(C₁-C₆ alkyl), —NHC(O)—(C₁-C₆        alkenyl), —NHC(O)-(aryl), —NHC(O)-(cycloalkyl),        —NHC(O)-(heterocyclyl), —NHC(O)—(C₁-C₆ alkyl)aryl,        —NHC(O)—(C₁-C₆ alkyl)cycloalkyl, —NHC(O)—(C₁-C₆        alkyl)heterocyclyl, —NHC(O)—(C₁-C₆ alkyl)amino, —NHC(O)—(C₁-C₆        alkyl)alkylamine, —NHC(O)—(C₁-C₆ alkyl)dialkylamine,        —NHC(O)—(C₁-C₆ alkyl)C(O)amino, —NHC(O)—(C₁-C₆        alkyl)C(O)alkylamine, —NHC(O)—(C₁-C₆ alkyl)C(O)dialkylamine,        —NHC(O)—(C₁-C₆ alkyl)N(H)—(C₁-C₆ alkyl)C(O)₂—(C₁-C₆ alkyl),        —NH—(C₁-C₆ alkyl)-C(O)-amino, —NH—(C₁-C₆ alkyl)-C(O)alkylamino,        —NH—(C₁-C₆ alkyl)-C(O)-dialkylamino, —NHC(O)—(C₁-C₆        alkyl)S(O)₂(C₁-C₆ alkyl), —NHC(O)—(C₁-C₆        alkyl)-S-(heterocyclyl), —NHS(O)₂—(C₁-C₆ alkyl),        —NHS(O)₂-(aryl), —NH—(C₁-C₆ alkyl)-S(O)₂-amino, —NH—(C₁-C₆        alkyl)-S(O)₂-alkylamino, —NH—(C₁-C₆ alkyl)-S(O)₂-dialkylamino,        —NHS(O)₂-(cycloalkyl), —NHS(O)₂-(heterocyclyl), —NHS(O)(C₁-C₆        alkyl), —NHS(O)(aryl), —NHS(O)(cycloalkyl),        —NHS(O)(heterocyclyl), —NHS(C₁-C₆ alkyl), —NHS(aryl),        —NHS(cycloalkyl), and —NH—S-(heterocyclyl),

wherein each of the above substituents can be further optionallysubstituted by 1-5 substituents selected from

-   -   amino,    -   C₁-C₆ alkylamine, C₁-C₆ dialkylamine,    -   C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ alkenyl, C₁-C₆ hydroxyl, and        C₁-C₆ hydroxyalkyl, each optionally substituted by    -   cyano,    -   halo, and    -   nitro,        or a pharmaceutically acceptable salt, hydrate, or stereoisomer        thereof.

In one embodiment, the invention relates to compounds of Formula I,wherein R² is NH₂.

In another embodiment, the invention relates to compounds of Formula I,wherein R³ is H.

In another embodiment, the invention relates to compounds of Formula I,wherein X is O and Y is S.

In another embodiment, the invention relates to compounds of Formula I,wherein R¹ is selected from

In another embodiment, the invention relates to compounds of the FormulaI selected from

In a another general aspect, the invention relates to 3,5-disubstitutedand 3,5,7-trisubstituted-3H-oxazolo and3H-thiazolo[4,5-d]pyrimidin-2-one compounds of Formula II

wherein

X is O or S, Y is O or S, Z is O or CH₂, R² is —NH₂, —NHC(O)R⁴, —NHR⁵,—N═CHNR⁶R⁷,

R⁴ is —C₁-C₇-alkyl or —O(C₁-C₇-alkyl),R⁵ is —C₁-C₇-alkyl,R⁶ and R⁷ are independently —C₁-C₇-alkyl or together with nitrogen forma 5- or 6-membered heterocyclic ring,

R¹³ is OH or SH,

R¹⁴ is H, —CH₂OH, or —CH₂—O—C(O)C₁₋₁₈alkyl,R¹⁵ is OH, alkenyl, —OC(O)C₁₋₁₈alkyl, —OC(O)aryl, or —OC(O)heterocyclyl,R¹⁶, R¹⁷, R¹⁸, and R¹⁹ are independently H, halo, N₃, alkyl,—(CH₂)_(m)OR²⁰, —(CH₂)_(m)OC(O)C₁₋₁₈alkyl, —OC(O)aryl, —OS(O)₂aryl, orR¹⁶ and R¹⁷ are an alkenyl, or R¹⁷ and R¹⁹ combine together to form adioxole ring,R²⁰ is H or alkyl,m is 0 or 1,n is 1 or 2,wherein if R² is NH₂, then one of the following must be present:

Z is CH₂;

either n is 2 or m is 1;at least one of R¹⁶, R¹⁷, R¹⁸, and R¹⁹ is halo, N₃, alkyl, or—(CH₂)_(m)OR²⁰ wherein m is 1, and wherein if R¹⁷ is N₃, then R¹⁸ andR¹⁹ are not H, and wherein if R¹⁷ is OH and R¹⁶ and R¹⁹ are H, then R¹⁸is not F; orR¹⁶ and R¹⁷ are an alkenyl,wherein the above alkyl, aryl, cycloalkyl, or heterocyclyl moieties areoptionally substituted by 1-4 substituents selected from

-   -   hydrogen,    -   alkanoyl,    -   alkylamine,    -   amino,    -   aryl, cycloalkyl, heterocyclyl,    -   azido,    -   C₁-C₆ alkyl, C₁-C₆ haloalkyl, C₁-C₆ hydroxyalkyl, C₁-C₆ alkoxy,        C₁-C₆ alkylamine, C₁-C₆ dialkylamine, C₂-C₆ alkenyl, or C₂-C₆        alkynyl, wherein each of which may be interrupted by one or more        hetero atoms,    -   carboxyl,    -   cyano,    -   halo,    -   hydroxy,    -   mercapto,    -   nitro,    -   thioalkyl,    -   —N═N—NH₂,    -   —C(O)₂—(C₁-C₆ alkyl), —C(O)₂-(aryl), —C(O)₂-(cycloalkyl),        —C(O)₂-(heterocyclyl), —O—(C₁-C₆ haloalkyl), —O—(C₁-C₆        alkyl)aryl, —O—(C₁-C₆ alkyl)cycloalkyl, —O—(C₁-C₆        alkyl)heterocyclyl, —O—(C₁-C₆ alkyl)amino, —O—(C₁-C₆        alkyl)alkylamino, —O—(C₁-C₆ alkyl)dialkylamino, —O—(C₁-C₆        alkyl)-C(O)-amino, —O—(C₁-C₆ alkyl)-C(O)-alkylamino, —O—(C₁-C₆        alkyl)-S(O)₂-amino, —O—(C₁-C₆ alkyl)-S(O)₂-alkylamino, —O—(C₁-C₆        alkyl)-S(O)₂-dialkylamino, —O—(C₁-C₆ alkyl)-C(O)-dialkylamino,        —O-aryl, —O-heterocyclyl, —NHC(O)—(C₁-C₆ alkyl), —NHC(O)—(C₁-C₆        alkenyl), —NHC(O)-(aryl), —NHC(O)-(cycloalkyl),        —NHC(O)-(heterocyclyl), —NHC(O)—(C₁-C₆ alkyl)aryl,        —NHC(O)—(C₁-C₆ alkyl)cycloalkyl, —NHC(O)—(C₁-C₆        alkyl)heterocyclyl, —NHC(O)—(C₁-C₆ alkyl)amino, —NHC(O)—(C₁-C₆        alkyl)alkylamine, —NHC(O)—(C₁-C₆ alkyl)dialkylamine,        —NHC(O)—(C₁-C₆ alkyl)C(O)amino, —NHC(O)—(C₁-C₆        alkyl)C(O)alkylamine, —NHC(O)—(C₁-C₆ alkyl)C(O)dialkylamine,        —NHC(O)—(C₁-C₆ alkyl)N(H)—(C₁-C₆ alkyl)C(O)₂—(C₁-C₆ alkyl),        —NH—(C₁-C₆ alkyl)-C(O)-amino, —NH—(C₁-C₆ alkyl)-C(O)-alkylamino,        —NH—(C₁-C₆ alkyl)-C(O)-dialkylamino, —NHC(O)—(C₁-C₆        alkyl)S(O)₂(C₁-C₆ alkyl), —NHC(O)—(C₁-C₆        alkyl)-S-(heterocyclyl), —NHS(O)₂—(C₁-C₆ alkyl),        —NHS(O)₂-(aryl), —NH—(C₁-C₆ alkyl)-S(O)₂-amino, —NH—(C₁-C₆        alkyl)-S(O)₂-alkylamino, —NH—(C₁-C₆ alkyl)-S(O)₂-dialkylamino,        —NHS(O)₂-(cycloalkyl), —NHS(O)₂-(heterocyclyl), —NHS(O)(C₁-C₆        alkyl), —NHS(O)(aryl), —NHS(O)(cycloalkyl),        —NHS(O)(heterocyclyl), —NHS(C₁-C₆ alkyl), —NHS(aryl),        —NHS(cycloalkyl), and —NH—S-(heterocyclyl),

wherein each of the above substituents can be further optionallysubstituted by 1-5 substituents selected from

-   -   amino,    -   C₁-C₆ alkylamine, C₁-C₆ dialkylamine,    -   C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ alkenyl, C₁-C₆ hydroxyl, and        C₁-C₆ hydroxyalkyl, each optionally substituted by    -   cyano,    -   halo, and    -   nitro,        or a pharmaceutically acceptable salt, hydrate, or stereoisomer        thereof.

In another embodiment, the invention relates to compounds of Formula II,wherein R² is NH₂.

In another embodiment, the invention relates to compounds of Formula II,wherein R¹³ is OH.

In another embodiment, the invention relates to compounds of Formula II,wherein X is O and Y is S.

In another embodiment, the invention relates to compounds of Formula IIselected from

In a another general aspect, the invention relates to 3,5-disubstitutedand 3,5,7-trisubstituted-3H-oxazolo and3H-thiazolo[4,5-d]pyrimidin-2-one compounds selected from

The invention is also directed to pharmaceutically active metabolites,pharmaceutically acceptable salts, and pharmaceutically acceptablesolvates of the compounds or metabolites of the Formula I prodrugs,Formula II compounds, and other compounds of the invention. Advantageousmethods of making the compounds of the invention are also described.

The Formula I prodrugs, Formula II compounds, and other compounds of theinvention are useful as immune system enhancers and have certain immunesystem properties including modulation, mitogenicity, augmentation,and/or potentiation or they are intermediates for compounds that havethese properties. The compounds are expected to express effects on atleast the natural killer, macrophages, dendritic or lymphocyte cells ofthe immune system of a host. Because of these properties they are usefulas antiviral and antitumor agents or as intermediates for antiviral andantitumor agents. They can be used to treat an affected host by servingas the active ingredients of suitable pharmaceutical compositions.

In one aspect of the invention, Formula I prodrugs, Formula IIcompounds, and other compounds of the invention are utilized to treatthe full range of viral diseases in mammals, including humans, byadministering to the mammal a therapeutically effective amount of thecompounds. Viral diseases contemplated to be treated with compounds ofthe invention include acute and chronic infections caused by both RNAand DNA viruses. Without limiting in any way the range of viralinfections that may be treated, Formula I prodrugs, Formula IIcompounds, and other compounds of the invention are particularly usefulin the treatment of infections caused by adenovirus, cytomegalovirus,hepatitis A virus (HAV), hepatitis B virus (HBV), flaviviruses includingYellow Fever virus and hepatitis C virus (HCV), herpes simplex type 1and 2, herpes zoster, human herpesvirus 6, human immunodeficiency virus(HIV), human papilloma virus (HPV), influenza A virus, influenza Bvirus, measles, parainfluenza virus, poliovirus, poxvirus (includingsmallpox and monkeypox virus), rhinovirus, respiratory syncytial virus(RSV), multiple families of viruses that cause hemorrhagic fevers,including the Arenaviruses (LCM, Junin virus, Machup virus, Guanaritovirus, and Lassa Fever), the Bunyaviruses (Hanta viruses and Rift ValleyFever) and Filoviruses (Ebola and Marburg virus), a range of viralencephalitides including West Nile virus, LaCrosse virus, CaliforniaEncephalitis virus, Venezuelan Equine Encephalitis virus, Eastern EquineEncephalitis virus, Western Equine Encephalitis virus, JapaneseEncephalitis virus, Kysanur Forest virus, and tickborne viruses such asCrimean-Congo Hemorrhagic fever virus.

In another aspect of the invention, Formula I prodrugs, Formula IIcompounds, and other compounds of the invention are utilized to treatbacterial, fungal, and protozoal infections in mammals by administeringto the mammal a therapeutically effective amount of the compounds. Thefull range of pathogenic microorganisms is contemplated to be treatableby the compounds of the present invention, including without limitationthose organisms that are resistant to antibiotics. The ability ofcompounds to activate multiple components of the immune system bypassesresistance mechanisms commonly found to reduce susceptibility toantibiotics, and thus treatment of infections in a mammal caused by suchresistant microorganisms by Formula I prodrugs, Formula II compounds,and other compounds of the invention is a particular utility of thepresent invention.

In another aspect of the invention, Formula I prodrugs, Formula IIcompounds, and other compounds of the invention are utilized to treattumors in mammals by administering to the mammal a therapeuticallyeffective amount of the compounds. Tumors or cancers contemplated to betreated include but are not limited to those caused by virus, and theeffect may involve inhibiting the transformation of virus-infected cellsto a neoplastic state, inhibiting the spread of viruses from transformedcells to other normal cells, and/or arresting the growth ofvirus-transformed cells. The compounds of the invention are expected tobe useful against a broad spectrum of tumors including but not limitedto carcinomas, sarcomas, and leukemias. Included in such a class aremammary, colon, bladder, lung, prostate, stomach, and pancreascarcinomas and lymphoblastic and myeloid leukemias.

In another aspect of the invention, a method of treating a mammalcomprises administering a therapeutically and/or prophylacticallyeffective amount of a pharmaceutical containing a compound of theinvention. In this aspect the effect may relate to modulation of someportion of the mammal's immune system, especially modulation of cytokineactivities of Th1 and Th2, including but not restricted to theinterleukin family, e.g., IL-1 through IL-12, and other cytokines suchas TNF alpha, and interferons including interferon alpha, interferonbeta, and interferon gamma, and their downstream effectors. Wheremodulation of Th1 and Th2 cytokines occurs, it is contemplated that themodulation may include stimulation of both Th1 and Th2, suppression ofboth Th1 and Th2, stimulation of either Th1 or Th2, and suppression ofthe other, or a bimodal modulation in which one effect on Th1/Th2 levels(such as generalized suppression) occurs at a high concentration, whileanother effect (such as stimulation of either Th1 or Th2 and suppressionof the other) occurs at a lower concentration.

In another aspect of the invention, pharmaceutical compositionscontaining a Formula I prodrug, Formula II compound, or other compoundof the invention are administered in a therapeutically effective dose toa mammal that is receiving anti-infective drugs not included in thecompounds of the invention. In a preferred aspect of this invention, thepharmaceutical compositions containing a Formula I prodrug, Formula IIcompound, or other compound of the invention are administered in atherapeutically effective dose with anti-infective drug(s) that actdirectly upon the infectious agent to inhibit the growth of or kill theinfectious agent.

In another aspect, the invention encompasses a method for treating orpreventing hepatitis C virus infection in a mammal in need thereof,preferably in a human in need thereof.

In another aspect, the invention encompasses a method for treating orpreventing hepatitis C virus infection in a patient in need thereof,comprising administering to the patient a therapeutically orprophylactically effective amount of a Formula I prodrug, Formula IIcompound, or other compound of the invention and a pharmaceuticallyacceptable excipient, carrier, or vehicle.

In a another aspect, the invention encompasses a method for treating orpreventing hepatitis C virus infection in a patient in need thereof,comprising administering to the patient a therapeutically orprophylactically effective amount of a compound of a Formula I prodrug,Formula II compound, or other compound of the invention and anadditional therapeutic agent, preferably an additional antiviral agentor anti-tumor agent as appropriate for the intended use.

In a preferred aspect of the invention, a pharmaceutical compositioncomprising a therapeutically effective amount of a Formula I prodrugprovides for improved oral availability and administration as animmunomodulator. In another preferred aspect of the invention, apharmaceutical composition comprising a therapeutically effective amountof a Formula I prodrug of the invention provides for masking the activestructure as the agent passes through lymphoid tissue lining thestomach, thereby minimizing activation of this tissue and allowing forimproved oral tolerability.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a plot of pg/ml IFN-α induced in human PBMCs from compound134 vs. pg/ml IFN-α induced by an identical concentration ofisatoribine.

FIG. 2 shows a plot of pg/ml IFN-α induced in human PBMCs from compound122 vs. pg/ml IFN-α induced by an identical concentration ofisatoribine.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS

Where the following terms are used in this specification, they are usedas defined below:

The terms “comprising” and “including” are used herein in their open,non-limiting sense.

The term “pyrimidine” refers to nitrogenous monocyclic heterocycles.

The term “alkyl”, as used herein, unless otherwise indicated, includessaturated monovalent hydrocarbon radicals having straight, branched, orcyclic moieties (including fused and bridged bicyclic and spirocyclicmoieties), or a combination of the foregoing moieties. For an alkylgroup to have cyclic moieties, the group must have at least three carbonatoms.

The term “alkenyl”, as used herein, unless otherwise indicated, includesalkyl moieties having at least one carbon-carbon double bond whereinalkyl is as defined above and including E and Z isomers of said alkenylmoiety.

The term “alkynyl”, as used herein, unless otherwise indicated, includesalkyl moieties having at least one carbon-carbon triple bond whereinalkyl is as defined above.

The term “alkoxy”, as used herein, unless otherwise indicated, includesO-alkyl groups wherein alkyl is as defined above.

The term “Me” means methyl, “Et” means ethyl, “Ac” means acetyl, “Bz”means benzoyl, and “Tol” means toluoyl.

The term “cycloalkyl”, as used herein, unless otherwise indicated refersto a non-aromatic, saturated or partially saturated, monocyclic orfused, spiro or unfused bicyclic or tricyclic hydrocarbon referred toherein containing a total of from 3 to 10 carbon atoms, preferably 5-8ring carbon atoms. Exemplary cycloalkyls include monocyclic rings havingfrom 3-7, preferably 3-6, carbon atoms, such as cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloheptyl and the like. Illustrative examplesof cycloalkyl are derived from, but not limited to, the following:

The term “aryl”, as used herein, unless otherwise indicated, includes anorganic radical derived from an aromatic hydrocarbon by removal of onehydrogen, such as phenyl or naphthyl.

The term “heterocyclyl” or “heterocyclic”, as used herein, unlessotherwise indicated, includes aromatic (e.g., a heteroaryl) andnon-aromatic heterocyclic groups containing one to four heteroatoms eachselected from O, S and N, wherein each heterocyclic group has from 4-10atoms in its ring system, and with the proviso that the ring of saidgroup does not contain two adjacent O or S atoms. Non-aromaticheterocyclic groups include groups having only 4 atoms in their ringsystem, but aromatic heterocyclic groups must have at least 5 atoms intheir ring system. The heterocyclic groups include benzo-fused ringsystems. An example of a 4 membered heterocyclic group is azetidinyl(derived from azetidine). An example of a 5 membered heterocyclic groupis thiazolyl and an example of a 10 membered heterocyclic group isquinolinyl. Examples of non-aromatic heterocyclic groups arepyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl,tetrahydropyranyl, dihydropyranyl, tetrahydrothiopyranyl, piperidino,morpholino, thiomorpholino, thioxanyl, piperazinyl, azetidinyl,oxetanyl, thietanyl, homopiperidinyl, oxepanyl, thiepanyl, oxazepinyl,diazepinyl, thiazepinyl, 1,2,3,6-tetrahydropyridinyl, 2-pyrrolinyl,3-pyrrolinyl, indolinyl, 2H-pyranyl, 4H-pyranyl, dioxanyl,1,3-dioxolanyl, pyrazolinyl, dithianyl, dithiolanyl, dihydropyranyl,dihydrothienyl, dihydrofuranyl, pyrazolidinyl, imidazolinyl,imidazolidinyl, 3-azabicyclo[3.1.0]hexanyl, 3-azabicyclo[4.1.0]heptanyl,3H-indolyl and quinolizinyl. Examples of aromatic heterocyclic groupsare pyridinyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl,tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl,isothiazolyl, pyrrolyl, quinolinyl, isoquinolinyl, indolyl,benzimidazolyl, benzofuranyl, cinnolinyl, indazolyl, indolizinyl,phthalazinyl, pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyl,oxadiazolyl, thiadiazolyl, furazanyl, benzofurazanyl, benzothiophenyl,benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl,naphthyridinyl, and furopyridinyl. The foregoing groups, as derived fromthe groups listed above, may be C-attached or N-attached where such ispossible. For instance, a group derived from pyrrole may be pyrrol-1-yl(N-attached) or pyrrol-3-yl (C-attached). Further, a group derived fromimidazole may be imidazol-1-yl (N-attached) or imidazol-3-yl(C-attached). The 4-10 membered heterocyclic may be optionallysubstituted on any ring carbon, sulfur, or nitrogen atom(s) by one totwo oxo, per ring. An example of a heterocyclic group wherein 2 ringcarbon atoms are substituted with oxo moieties is1,1-dioxo-thiomorpholinyl. Other illustrative examples of 4-10 memberedheterocyclic are derived from, but not limited to, the following:

The term “immunomodulator” refers to natural or synthetic productscapable of modifying the normal or aberrant immune system throughstimulation or suppression.

The term “preventing” refers to the ability of a compound or compositionof the invention to prevent a disease identified herein in patientsdiagnosed as having the disease or who are at risk of developing suchdisease. The term also encompasses preventing further progression of thedisease in patients who are already suffering from or have symptoms ofsuch disease.

The term “patient” or “subject” means an animal (e.g., cow, horse,sheep, pig, chicken, turkey, quail, cat, dog, mouse, rat, rabbit, guineapig, etc.) or a mammal, including chimeric and transgenic animals andmammals. In the treatment or prevention of HCV infection, the term“patient” or “subject” preferably means a monkey or a human, mostpreferably a human. In a specific embodiment the patient or subject isinfected by or exposed to the hepatitis C virus. In certain embodiments,the patient is a human infant (age 0-2), child (age 2-17), adolescent(age 12-17), adult (age 18 and up) or geriatric (age 70 and up) patient.In addition, the patient includes immunocompromised patients such as HIVpositive patients, cancer patients, patients undergoing immunotherapy orchemotherapy. In a particular embodiment, the patient is a healthyindividual, i.e., not displaying symptoms of other viral infections.

The term a “therapeutically effective amount” refers to an amount of thecompound of the invention sufficient to provide a benefit in thetreatment or prevention of viral disease, to delay or minimize symptomsassociated with viral infection or viral-induced disease, or to cure orameliorate the disease or infection or cause thereof. In particular, atherapeutically effective amount means an amount sufficient to provide atherapeutic benefit in vivo. Used in connection with an amount of acompound of the invention, the term preferably encompasses a non-toxicamount that improves overall therapy, reduces or avoids symptoms orcauses of disease, or enhances the therapeutic efficacy of or synergieswith another therapeutic agent.

The term a “prophylactically effective amount” refers to an amount of acompound of the invention or other active ingredient sufficient toresult in the prevention of infection, recurrence or spread of viralinfection. A prophylactically effective amount may refer to an amountsufficient to prevent initial infection or the recurrence or spread ofthe infection or a disease associated with the infection. Used inconnection with an amount of a compound of the invention, the termpreferably encompasses a non-toxic amount that improves overallprophylaxis or enhances the prophylactic efficacy of or synergies withanother prophylactic or therapeutic agent.

The term “in combination” refers to the use of more than oneprophylactic and/or therapeutic agents simultaneously or sequentiallyand in a manner that their respective effects are additive orsynergistic.

The term “treating” refers to:

(i) preventing a disease, disorder, or condition from occurring in ananimal that may be predisposed to the disease, disorder and/orcondition, but has not yet been diagnosed as having it;

(ii) inhibiting the disease, disorder, or condition, i.e., arresting itsdevelopment; and

(iii) relieving the disease, disorder, or condition, i.e., causingregression of the disease, disorder, and/or condition.

The terms “α” and “β” indicate the specific stereochemical configurationof a substituent at an asymmetric carbon atom in a chemical structure asdrawn.

The compounds of the invention may exhibit the phenomenon oftautomerism. While the formula drawings cannot expressly depict allpossible tautomeric forms, it is to be understood they are intended torepresent any tautomeric form of the depicted compound and are not to belimited merely to a specific compound form depicted by the formuladrawings. For example, it is understood for Formula II that regardlessof whether or not the substituents are shown in their enol or their ketoform, they represent the same compound (as shown in the example below).

Some of the inventive compounds may exist as single stereoisomers (i.e.,essentially free of other stereoisomers), racemates, and/or mixtures ofenantiomers and/or diastereomers. All such single stereoisomers,racemates and mixtures thereof are intended to be within the scope ofthe present invention. Preferably, the inventive compounds that areoptically active are used in optically pure form.

As generally understood by those skilled in the art, an optically purecompound having one chiral center (i.e., one asymmetric carbon atom) isone that consists essentially of one of the two possible enantiomers(i.e., is enantiomerically pure), and an optically pure compound havingmore than one chiral center is one that is both diastereomerically pureand enantiomerically pure. Preferably, the compounds of the presentinvention are used in a form that is at least 90% optically pure, thatis, a form that contains at least 90% of a single isomer (80%enantiomeric excess (“e.e.”) or diastereomeric excess (“d.e.”)), morepreferably at least 95% (90% e.e. or d.e.), even more preferably atleast 97.5% (95% e.e. or d.e.), and most preferably at least 99% (98%e.e. or d.e.).

Additionally, Formula I prodrugs, Formula II compounds, and othercompounds of the invention are intended to cover solvated as well asunsolvated forms of the identified structures. For example, Formula Iincludes compounds of the indicated structure in both hydrated andnon-hydrated forms. Other examples of solvates include the structures incombination with isopropanol, ethanol, methanol, DMSO, ethyl acetate,acetic acid, or ethanolamine.

In addition to Formula I prodrugs, Formula II compounds, and othercompounds of the invention, the invention includes pharmaceuticallyactive metabolites and pharmaceutically acceptable salts of suchcompounds and metabolites.

“A pharmaceutically acceptable prodrug” is a compound that may beconverted under physiological conditions or by solvolysis to thespecified compound or to a pharmaceutically acceptable salt of suchcompound prior to exhibiting its pharmacological effect(s). Typically,the prodrug is formulated with the objective(s) of improved chemicalstability, improved patient acceptance and compliance, improvedbioavailability, prolonged duration of action, improved organselectivity, improved formulation (e.g., increased hydrosolubility),and/or decreased side effects (e.g., toxicity). The prodrug can bereadily prepared using methods known in the art, such as those describedby Burger's Medicinal Chemistry and Drug Chemistry, 1, 172-178, 949-982(1995). See also Bertolini et al., J. Med. Chem., 40, 2011-2016 (1997);Shan, et al., J. Pharm. Sci., 86 (7), 765-767; Bagshawe, Drug Dev. Res.,34, 220-230 (1995); Bodor, Advances in Drug Res., 13, 224-331 (1984);Bundgaard, Design of Prodrugs (Elsevier Press 1985); Larsen, Design andApplication of Prodrugs, Drug Design and Development (Krogsgaard-Larsenet al., eds., Harwood Academic Publishers, 1991); Dear et al., J.Chromatogr. B, 748, 281-293 (2000); Spraul et al., J. Pharmaceutical &Biomedical Analysis, 10, 601-605 (1992); and Prox et al., Xenobiol., 3,103-112 (1992).

“A pharmaceutically active metabolite” is intended to mean apharmacologically active product produced through metabolism in the bodyof a specified compound or salt thereof. After entry into the body, mostdrugs are substrates for chemical reactions that may change theirphysical properties and biologic effects. These metabolic conversions,which usually affect the polarity of the compounds of the invention,alter the way in which drugs are distributed in and excreted from thebody. However, in some cases, metabolism of a drug is required fortherapeutic effect. For example, anticancer drugs of the anti-metaboliteclass must be converted to their active forms after they have beentransported into a cancer cell.

Since most drugs undergo metabolic transformation of some kind, thebiochemical reactions that play a role in drug metabolism may benumerous and diverse. The main site of drug metabolism is the liver,although other tissues may also participate.

A feature characteristic of many of these transformations is that themetabolic products, or “metabolites,” are more polar than the parentdrugs, although a polar drug does sometime yield a less polar product.Substances with high lipid/water partition coefficients, which passeasily across membranes, also diffuse back readily from tubular urinethrough the renal tubular cells into the plasma. Thus, such substancestend to have a low renal clearance and a long persistence in the body.If a drug is metabolized to a more polar compound, one with a lowerpartition coefficient, its tubular reabsorption will be greatly reduced.Moreover, the specific secretory mechanisms for anions and cations inthe proximal renal tubules and in the parenchymal liver cells operateupon highly polar substances.

As a specific example, phenacetin (acetophenetidin) and acetanilide areboth mild analgesic and antipyretic agents, but are transformed withinthe body to a more polar and more effective metabolite,p-hydroxyacetanilid (acetaminophen), which is widely used today. When adose of acetanilide is given to a person, the successive metabolitespeak and decay in the plasma sequentially. During the first hour,acetanilide is the principal plasma component. In the second hour, asthe acetanilide level falls, the metabolite acetaminophen concentrationreaches a peak. Finally, after a few hours, the principal plasmacomponent is a further metabolite that is inert and can be excreted fromthe body. Thus, the plasma concentrations of one or more metabolites, aswell as the drug itself, can be pharmacologically important.

“A pharmaceutically acceptable salt” is intended to mean a salt thatretains the biological effectiveness of the free acids and bases of thespecified compound and that is not biologically or otherwiseundesirable. A compound of the invention may possess a sufficientlyacidic, a sufficiently basic, or both functional groups, and accordinglyreact with any of a number of inorganic or organic bases, and inorganicand organic acids, to form a pharmaceutically acceptable salt. Exemplarypharmaceutically acceptable salts include those salts prepared byreaction of the compounds of the present invention with a mineral ororganic acid or an inorganic base, such as salts including sulfates,pyrosulfates, bisulfates, sulfites, bisulfites, phosphates,monohydrogenphosphates, dihydrogenphosphates, metaphosphates,pyrophosphates, chlorides, bromides, iodides, acetates, propionates,decanoates, caprylates, acrylates, formates, isobutyrates, caproates,heptanoates, propiolates, oxalates, malonates, succinates, suberates,sebacates, fumarates, maleates, butyne-1,4-dioates, hexyne-1,6-dioates,benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates,hydroxybenzoates, methoxybenzoates, phthalates, sulfonates,xylenesulfonates, phenylacetates, phenylpropionates, phenylbutyrates,citrates, lactates, γ-hydroxybutyrates, glycolates, tartrates,methane-sulfonates, propanesulfonates, naphthalene-1-sulfonates,naphthalene-2-sulfonates, and mandelates.

If the inventive compound is a base, the desired pharmaceuticallyacceptable salt may be prepared by any suitable method available in theart, for example, treatment of the free base with an inorganic acid,such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid and the like, or with an organic acid, such as aceticacid, maleic acid, succinic acid, mandelic acid, fumaric acid, malonicacid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, apyranosidyl acid, such as glucuronic acid or galacturonic acid, analpha-hydroxy acid, such as citric acid or tartaric acid, an amino acid,such as aspartic acid or glutamic acid, an aromatic acid, such asbenzoic acid or cinnamic acid, a sulfonic acid, such asp-toluenesulfonic acid or ethanesulfonic acid, or the like.

If the inventive compound is an acid, the desired pharmaceuticallyacceptable salt may be prepared by any suitable method, for example,treatment of the free acid with an inorganic or organic base, such as anamine (primary, secondary or tertiary), an alkali metal hydroxide oralkaline earth metal hydroxide, or the like. Illustrative examples ofsuitable salts include organic salts derived from amino acids, such asglycine and arginine, ammonia, primary, secondary, and tertiary amines,and cyclic amines, such as piperidine, morpholine and piperazine, andinorganic salts derived from sodium, calcium, potassium, magnesium,manganese, iron, copper, zinc, aluminum and lithium.

In the case of agents that are solids, it is understood by those skilledin the art that the inventive compounds and salts may exist in differentcrystal or polymorphic forms, all of which are intended to be within thescope of the present invention and specified formulas.

Methods of Treatment and Prevention of Hepatitis C Viral Infections

The present invention provides methods for treating or preventing ahepatitis C virus infection in a patient in need thereof.

The present invention further provides methods for introducing atherapeutically effective amount of a Formula I prodrug, Formula IIcompound, or other compound of the invention or combination of suchcompounds into the blood stream of a patient in the treatment and/orprevention of hepatitis C viral infections.

The magnitude of a prophylactic or therapeutic dose of a Formula Iprodrug, Formula II compound, or other compound of the invention or apharmaceutically acceptable salt, solvate, or hydrate, thereof in theacute or chronic treatment or prevention of an infection will vary,however, with the nature and severity of the infection, and the route bywhich the active ingredient is administered. The dose, and in some casesthe dose frequency, will also vary according to the infection to betreated, the age, body weight, and response of the individual patient.Suitable dosing regimens can be readily selected by those skilled in theart with due consideration of such factors.

The methods of the present invention are particularly well suited forhuman patients. In particular, the methods and doses of the presentinvention can be useful for immunocompromised patients including, butnot limited to cancer patients, HIV infected patients, and patients withan immunodegenerative disease. Furthermore, the methods can be usefulfor immunocompromised patients currently in a state of remission. Themethods and doses of the present invention are also useful for patientsundergoing other antiviral treatments. The prevention methods of thepresent invention are particularly useful for patients at risk of viralinfection. These patients include, but are not limited to health careworkers, e.g., doctors, nurses, hospice care givers; military personnel;teachers; childcare workers; patients traveling to, or living in,foreign locales, in particular third world locales including social aidworkers, missionaries, and foreign diplomats. Finally, the methods andcompositions include the treatment of refractory patients or patientsresistant to treatment such as resistance to reverse transcriptaseinhibitors, protease inhibitors, etc.

Doses

Toxicity and efficacy of the compounds of the invention can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD₅₀ (the dose lethal to50% of the population) and the ED₅₀ (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratioLD₅₀/ED₅₀.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage of the compounds for use inhumans. The dosage of such compounds lie preferably within a range ofcirculating concentrations that include the ED₅₀ with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. For anycompound used in the method of the invention, the therapeuticallyeffective dose can be estimated initially from cell culture assays. Adose may be formulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound that achieves a half-maximal inhibition of symptoms)as determined in cell culture; alternatively, the dose of the compoundsmay be formulated in animal models to achieve a circulating plasmaconcentration range of the compound that corresponds to theconcentration required to achieve a fixed magnitude of response. Suchinformation can be used to more accurately determine useful doses inhumans. Levels in plasma may be measured, for example, by highperformance liquid chromatography.

The protocols and compositions of the invention are preferably tested invitro, and then in vivo, for the desired therapeutic or prophylacticactivity, prior to use in humans. For example, in vitro assays which canbe used to determine whether administration of a specific therapeuticprotocol is indicated, include in vitro cell culture assays in whichcells that are responsive to the effects of Formula I prodrugs, FormulaII compounds, and other compounds of the invention are exposed to theligand and the magnitude of response is measured by an appropriatetechnique. The assessment of the compounds is then evaluated withrespect to the compound potency, and the degree of conversion betweenthe Formula I prodrug and Formula II parent compound. Compounds for usein methods of the invention can be tested in suitable animal modelsystems prior to testing in humans, including but not limited to inrats, mice, chicken, cows, monkeys, rabbits, hamsters, etc. Thecompounds can then be used in the appropriate clinical trials.

The magnitude of a prophylactic or therapeutic dose of a Formula Iprodrug, Formula II compound, or other compound of the invention or apharmaceutically acceptable salt, solvate, or hydrate thereof in theacute or chronic treatment or prevention of an infection or conditionwill vary with the nature and severity of the infection, and the routeby which the active ingredient is administered. The dose, and perhapsthe dose frequency, will also vary according to the infection to betreated, the age, body weight, and response of the individual patient.Suitable dosing regimens can be readily selected by those skilled in theart with due consideration of such factors. In one embodiment, the doseadministered depends upon the specific compound to be used, and theweight and condition of the patient. Also, the dose may differ forvarious particular compounds of the invention; suitable doses can bepredicted on the basis of the aforementioned in vitro measurements andon the basis of animal studies, such that smaller doses will be suitablefor those compounds that show effectiveness at lower concentrations thanother compounds when measured in the systems described or referencedherein. In general, the dose per day is in the range of from about 0.001to 100 mg/kg, preferably about 1 to 25 mg/kg, more preferably about 5 to15 mg/kg. For treatment of humans infected by hepatitis C viruses, about0.1 mg to about 15 g per day is administered in about one to fourdivisions a day, preferably 100 mg to 12 g per day, more preferably from100 mg to 8000 mg per day.

Additionally, the recommended daily dose ran can be administered incycles as single agents or in combination with other therapeutic agents.In one embodiment, the daily dose is administered in a single dose or inequally divided doses. In a related embodiment, the recommended dailydose can be administered once time per week, two times per week, threetimes per week, four times per week or five times per week.

In a preferred embodiment, the compounds of the invention areadministered to provide systemic distribution of the compound within thepatient. In a related embodiment, the compounds of the invention areadministered to produce a systemic effect in the body.

In another embodiment the compounds of the invention are administeredvia oral, mucosal (including sublingual, buccal, rectal, nasal, orvaginal), parenteral (including subcutaneous, intramuscular, bolusinjection, intraarterial, or intravenous), transdermal, or topicaladministration. In a specific embodiment the compounds of the inventionare administered via mucosal (including sublingual, buccal, rectal,nasal, or vaginal), parenteral (including subcutaneous, intramuscular,bolus injection, intraarterial, or intravenous), transdermal, or topicaladministration. In a further specific embodiment, the compounds of theinvention are administered via oral administration. In a furtherspecific embodiment, the compounds of the invention are not administeredvia oral administration.

Different therapeutically effective amounts may be applicable fordifferent infections, as will be readily known by those of ordinaryskill in the art. Similarly, amounts sufficient to treat or prevent suchinfections, but insufficient to cause, or sufficient to reduce, adverseeffects associated with conventional therapies are also encompassed bythe above described dosage amounts and dose frequency schedules.

Combination Therapy

Specific methods of the invention further comprise the administration ofan additional therapeutic agent (i.e., a therapeutic agent other than acompound of the invention). In certain embodiments of the presentinvention, the compounds of the invention can be used in combinationwith at least one other therapeutic agent. Therapeutic agents include,but are not limited to antibiotics, antiemetic agents, antidepressants,and antifungal agents, anti-inflammatory agents, antiviral agents,anticancer agents, immunomodulatory agents, β-interferons, alkylatingagents, hormones or cytokines. In a preferred embodiment the inventionencompasses the administration of an additional therapeutic agent thatis HCV specific or demonstrates anti-HCV activity.

The Formula I prodrugs, Formula II compounds, and other compounds of theinvention can be administered or formulated in combination withantibiotics. For example, they can be formulated with a macrolide (e.g.,tobramycin (Tobi®)), a cephalosporin (e.g., cephalexin (Keflex®),cephradine (Velosef®), cefuroxime (Ceftin®), cefprozil (Cefzil®),cefaclor (Ceclor®), cefixime (Suprax®) or cefadroxil (Duricef®)), aclarithromycin (e.g., clarithromycin (Biaxin®)), an erythromycin (e.g.,erythromycin (EMycin®)), a penicillin (e.g., penicillin V (V-Cillin K®or Pen Vee K®)) or a quinolone (e.g., ofloxacin (Floxin®), ciprofloxacin(Cipro®) or norfloxacin (Noroxin®)), aminoglycoside antibiotics (e.g.,apramycin, arbekacin, bambermycins, butirosin, dibekacin, neomycin,neomycin, undecylenate, netilmicin, paromomycin, ribostamycin,sisomicin, and spectinomycin), amphenicol antibiotics (e.g.,azidamfenicol, chloramphenicol, florfenicol, and thiamphenicol),ansamycin antibiotics (e.g., rifamide and rifampin), carbacephems (e.g.,loracarbef), carbapenems (e.g., biapenem and imipenem), cephalosporins(e.g., cefaclor, cefadroxil, cefamandole, cefatrizine, cefazedone,cefozopran, cefpimizole, cefpiramide, and cefpirome), cephamycins (e.g.,cefbuperazone, cefmetazole, and cefminox), monobactams (e.g., aztreonam,carumonam, and tigemonam), oxacephems (e.g., flomoxef, and moxalactam),penicillins (e.g., amdinocillin, amdinocillin pivoxil, amoxicillin,bacampicillin, benzylpenicillinic acid, benzylpenicillin sodium,epicillin, fenbenicillin, floxacillin, penamccillin, penethamatehydriodide, penicillin o-benethamine, penicillin 0, penicillin V,penicillin V benzathine, penicillin V hydrabamine, penimepicycline, andphencihicillin potassium), lincosamides (e.g., clindamycin, andlincomycin), amphomycin, bacitracin, capreomycin, colistin, enduracidin,enviomycin, tetracyclines (e.g., apicycline, chlortetracycline,clomocycline, and demeclocycline), 2,4-diaminopyrimidines (e.g.,brodimoprim), nitrofurans (e.g., furaltadone, and furazolium chloride),quinolones and analogs thereof (e.g., cinoxacin, clinafloxacin,flumequine, and grepagloxacin), sulfonamides (e.g., acetylsulfamethoxypyrazine, benzylsulfamide, noprylsulfamide,phthalylsulfacetamide, sulfachrysoidine, and sulfacytine), sulfones(e.g., diathymosulfone, glucosulfone sodium, and solasulfone),cycloserine, mupirocin and tuberin.

The Formula I prodrugs, Formula II compounds, and other compounds of theinvention can also be administered or formulated in combination with anantiemetic agent. Suitable antiemetic agents include, but are notlimited to, metoclopromide, domperidone, prochlorperazine, promethazine,chlorpromazine, trimethobenzamide, ondansetron, granisetron,hydroxyzine, acethylleucine monoethanolamine, alizapride, azasetron,benzquinamide, bietanautine, bromopride, buclizine, clebopride,cyclizine, dimenhydrinate, diphenidol, dolasetron, meclizine,methallatal, metopimazine, nabilone, oxyperndyl, pipamazine,scopolamine, sulpiride, tetrahydrocannabinols, thiethylperazine,thioproperazine, tropisetron, and mixtures thereof.

The Formula I prodrugs, Formula II compounds, and other compounds of theinvention can be administered or formulated in combination with anantidepressant. Suitable antidepressants include, but are not limitedto, binedaline, caroxazone, citalopram, dimethazan, fencamine,indalpine, indeloxazine hydrocholoride, nefopam, nomifensine,oxitriptan, oxypertine, paroxetine, sertraline, thiazesim, trazodone,benmoxine, iproclozide, iproniazid, isocarboxazid, nialamide, octamoxin,phenelzine, cotinine, rolicyprine, rolipram, maprotiline, metralindole,mianserin, mirtazepine, adinazolam, amitriptyline, amitriptylinoxide,amoxapine, butriptyline, clomipramine, demexiptiline, desipramine,dibenzepin, dimetacrine, dothiepin, doxepin, fluacizine, imipramine,imipramine N-oxide, iprindole, lofepramine, melitracen, metapramine,nortriptyline, noxiptilin, opipramol, pizotyline, propizepine,protriptyline, quinupramine, tianeptine, trimipramine, adrafinil,benactyzine, bupropion, butacetin, dioxadrol, duloxetine, etoperidone,febarbamate, femoxetine, fenpentadiol, fluoxetine, fluvoxamine,hematoporphyrin, hypericin, levophacetoperane, medifoxamine,milnacipran, minaprine, moclobemide, nefazodone, oxaflozane, piberaline,prolintane, pyrisuccideanol, ritanserin, roxindole, rubidium chloride,sulpiride, tandospirone, thozalinone, tofenacin, toloxatone,tranylcypromine, L-tryptophan, venlafaxine, viloxazine, and zimeldine.

The Formula I prodrugs, Formula II compounds, and other compounds of theinvention can be administered or formulated in combination with anantifungal agent. Suitable antifungal agents include but are not limitedto amphotericin B, itraconazole, ketoconazole, fluconazole, intrathecal,flucytosine, miconazole, butoconazole, clotrimazole, nystatin,terconazole, tioconazole, ciclopirox, econazole, haloprogrin, naftifine,terbinafine, undecylenate, and griseofuldin.

The Formula I prodrugs, Formula II compounds, and other compounds of theinvention can be administered or formulated in combination with ananti-inflammatory agent. Useful anti-inflammatory agents include, butare not limited to, non-steroidal anti-inflammatory drugs such assalicylic acid, acetylsalicylic acid, methyl salicylate, diflunisal,salsalate, olsalazine, sulfasalazine, acetaminophen, indomethacin,sulindac, etodolac, mefenamic acid, meclofenamate sodium, tolmetin,ketorolac, dichlofenac, ibuprofen, naproxen, naproxen sodium,fenoprofen, ketoprofen, flurbinprofen, oxaprozin, piroxicam, meloxicam,ampiroxicam, droxicam, pivoxicam, tenoxicam, nabumetome, phenylbutazone,oxyphenbutazone, antipyrine, aminopyrine, apazone and nimesulide;leukotriene antagonists including, but not limited to, zileuton,aurothioglucose, gold sodium thiomalate and auranofin; steroidsincluding, but not limited to, alclometasone diproprionate, amcinonide,beclomethasone dipropionate, betametasone, betamethasone benzoate,betamethasone diproprionate, betamethasone sodium phosphate,betamethasone valerate, clobetasol proprionate, clocortolone pivalate,hydrocortisone, hydrocortisone derivatives, desonide, desoximatasone,dexamethasone, flunisolide, flucoxinolide, flurandrenolide, halcinocide,medrysone, methylprednisolone, methprednisolone acetate,methylprednisolone sodium succinate, mometasone furoate, paramethasoneacetate, prednisolone, prednisolone acetate, prednisolone sodiumphosphate, prednisolone tebuatate, prednisone, triamcinolone,triamcinolone acetonide, triamcinolone diacetate, and triamcinolonehexacetonide; and other anti-inflammatory agents including, but notlimited to, methotrexate, colchicine, allopurinol, probenecid,sulfinpyrazone and benzbromarone.

The Formula I prodrugs, Formula II compounds, and other compounds of theinvention can be administered or formulated in combination with anotherantiviral agent. Useful antiviral agents include, but are not limitedto, protease inhibitors, nucleoside reverse transcriptase inhibitors,non-nucleoside reverse transcriptase inhibitors and nucleoside analogs.The antiviral agents include but are not limited to zidovudine,acyclovir, gangcyclovir, vidarabine, idoxuridine, trifluridine,levovirin, viramidine and ribavirin, as well as foscarnet, amantadine,rimantadine, saquinavir, indinavir, amprenavir, lopinavir, ritonavir,the alpha-interferons; beta-interferons; adefovir, clevadine, entecavir,pleconaril.

The Formula I prodrugs, Formula II compounds, and other compounds of theinvention can be administered or formulated in combination with animmunomodulatory agent. Immunomodulatory agents include, but are notlimited to, methothrexate, leflunomide, cyclophosphamide, cyclosporineA, mycophenolate mofetil, rapamycin (sirolimus), mizoribine,deoxyspergualin, brequinar, malononitriloamindes (e.g., leflunamide), Tcell receptor modulators, and cytokine receptor modulators, peptidemimetics, and antibodies (e.g., human, humanized, chimeric, monoclonal,polyclonal, Fvs, ScFvs, Fab or F(ab)2 fragments or epitope bindingfragments), nucleic acid molecules (e.g., antisense nucleic acidmolecules and triple helices), small molecules, organic compounds, andinorganic compounds. Examples of T cell receptor modulators include, butare not limited to, anti-T cell receptor antibodies (e.g., anti-CD4antibodies (e.g., cM-T412 (Boeringer), IDEC-CE9.1® (IDEC and SKB), mAB4162W94, Orthoclone and OKTcdr4a (Janssen-Cilag)), anti-CD3 antibodies(e.g., Nuvion (Product Design Labs), OKT3 (Johnson & Johnson), orRituxan (IDEC)), anti-CD5 antibodies (e.g., an anti-CD5 ricin-linkedimmunoconjugate), anti-CD7 antibodies (e.g., CHH-380 (Novartis)),anti-CD8 antibodies, anti-CD40 ligand monoclonal antibodies (e.g.,IDEC-131 (IDEC)), anti-CD52 antibodies (e.g., CAMPATH 1H (Ilex)),anti-CD2 antibodies, anti-CD11a antibodies (e.g., Xanelim (Genentech)),and anti-B7 antibodies (e.g., IDEC-114 (IDEC)) and CTLA4-immunoglobulin.Examples of cytokine receptor modulators include, but are not limitedto, soluble cytokine receptors (e.g., the extracellular domain of aTNF-α receptor or a fragment thereof, the extracellular domain of anIL-1β receptor or a fragment thereof, and the extracellular domain of anIL-6 receptor or a fragment thereof), cytokines or fragments thereof(e.g., interleukin (IL)-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9,IL-10, IL-11, IL-12, IL-15, TNF-α, interferon (IFN)-α, IFN-β, IFN-γ, andGM-CSF), anti-cytokine receptor antibodies (e.g., anti-IFN receptorantibodies, anti-IL-2 receptor antibodies (e.g., Zenapax (Protein DesignLabs)), anti-IL-4 receptor antibodies, anti-IL-6 receptor antibodies,anti-IL-10 receptor antibodies, and anti-IL-12 receptor antibodies),anti-cytokine antibodies (e.g., anti-IFN antibodies, anti-TNF-αantibodies, anti-IL-1β antibodies, anti-IL-6 antibodies, anti-IL-8antibodies (e.g., ABX-IL-8 (Abgenix)), and anti-IL-12 antibodies).

The Formula I prodrugs, Formula II compounds, and other compounds of theinvention can be administered or formulated in combination with an agentwhich inhibits viral enzymes, including but not limited to inhibitors ofHCV protease, such as BILN 2061 and inhibitors of NSSb polymerase suchas NM107 and its prodrug NM283 (Idenix Pharmaceuticals, Inc., Cambridge,Mass.).

The Formula I prodrugs, Formula II compounds, and other compounds of theinvention can be administered or formulated in combination with an agentwhich inhibits HCV polymerase such as those described in Wu, Curr DrugTargets Infect Disord. 2003; 3(3):207-19 or in combination withcompounds that inhibit the helicase function of the virus such as thosedescribed in Bretner M, et al Nucleosides Nucleotides Nucleic Acids.2003; 22(5-8):1531, or with inhibitors of other HCV specific targetssuch as those described in Zhang X. IDrugs. 2002; 5(2):154-8.

The Formula I prodrugs, Formula II compounds, and other compounds of theinvention can be administered or formulated in combination with an agentwhich inhibits viral replication.

The Formula I prodrugs, Formula II compounds, and other compounds of theinvention can be administered or formulated in combination withcytokines. Examples of cytokines include, but are not limited to,interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4),interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7),interleukin-9 (IL-9), interleukin-10 (IL-10), interleukin-12 (IL-12),interleukin 15 (IL-15), interleukin 18 (IL-18), platelet derived growthfactor (PDGF), erythropoietin (Epo), epidermal growth factor (EGF),fibroblast growth factor (FGF), granulocyte macrophage stimulatingfactor (GM-CSF), granulocyte colony stimulating factor (G-CSF),macrophage colony stimulating factor (M-CSF), prolactin, and interferon(IFN), e.g., IFN-alpha, and IFN-gamma).

The Formula I prodrugs, Formula II compounds, and other compounds of theinvention can be administered or formulated in combination withhormones. Examples of hormones include, but are not limited to,luteinizing hormone releasing hormone (LHRH), growth hormone (GH),growth hormone releasing hormone, ACTH, somatostatin, somatotropin,somatomedin, parathyroid hormone, hypothalamic releasing factors,insulin, glucagon, enkephalins, vasopressin, calcitonin, heparin, lowmolecular weight heparins, heparinoids, synthetic and natural opioids,insulin thyroid stimulating hormones, and endorphins.

The Formula I prodrugs, Formula II compounds, and other compounds of theinvention can be administered or formulated in combination withβ-interferons which include, but are not limited to, interferon beta-1a,interferon beta-1b.

The Formula I prodrugs, Formula II compounds, and other compounds of theinvention can be administered or formulated in combination withα-interferons which include, but are not limited to, interferon alpha-1,interferon alpha-2a (roferon), interferon alpha-2b, intron, Peg-Intron,Pegasys, consensus interferon (infergen) and albuferon.

The Formula I prodrugs, Formula II compounds, and other compounds of theinvention can be administered or formulated in combination with anabsorption enhancer, particularly those which target the lymphaticsystem, including, but not limited to sodium glycocholate; sodiumcaprate; N-lauryl-ÿ-D-maltopyranoside; EDTA; mixed micelle; and thosereported in Muranishi Crit. Rev. Ther. Drug Carrier Syst., 7-1-33, whichis hereby incorporated by reference in its entirety. Other knownabsorption enhancers can also be used. Thus, the invention alsoencompasses a pharmaceutical composition comprising one or more FormulaI prodrugs, Formula II compounds, and other compounds of the inventionand one or more absorption enhancers.

The Formula I prodrugs, Formula II compounds, and other compounds of theinvention can be administered or formulated in combination with analkylating agent. Examples of alkylating agents include, but are notlimited to nitrogen mustards, ethylenimines, methylmelamines, alkylsulfonates, nitrosoureas, triazenes, mechlorethamine, cyclophosphamide,ifosfamide, melphalan, chlorambucil, hexamethylmelaine, thiotepa,busulfan, carmustine, streptozocin, dacarbazine and temozolomide.

The Formula I prodrugs, Formula II compounds, and other compounds of theinvention and the other therapeutics agent can act additively or, morepreferably, synergistically. In a preferred embodiment, a compositioncomprising a compound of the invention is administered concurrently withthe administration of another therapeutic agent, which can be part ofthe same composition or in a different composition from that comprisingthe compounds of the invention. In another embodiment, a compound of theinvention is administered prior to or subsequent to administration ofanother therapeutic agent. In a separate embodiment, a compound of theinvention is administered to a patient who has not previously undergoneor is not currently undergoing treatment with another therapeutic agent,particularly an antiviral agent.

In one embodiment, the methods of the invention comprise theadministration of one or more Formula I prodrugs, Formula II compounds,and other compounds of the invention without an additional therapeuticagent.

Pharmaceutical Compositions and Dosage Forms

Pharmaceutical compositions and single unit dosage forms comprising aFormula I prodrugs, Formula II compounds, and other compounds of theinvention, or a pharmaceutically acceptable salt, or hydrate thereof,are also encompassed by the invention. Individual dosage forms of theinvention may be suitable for oral, mucosal (including sublingual,buccal, rectal, nasal, or vaginal), parenteral (including subcutaneous,intramuscular, bolus injection, intraarterial, or intravenous),transdermal, or topical administration. Pharmaceutical compositions anddosage forms of the invention typically also comprise one or morepharmaceutically acceptable excipients. Sterile dosage forms are alsocontemplated.

In an alternative embodiment, a pharmaceutical composition encompassedby this embodiment includes a Formula I prodrug, Formula II compound, orother compound of the invention, or a pharmaceutically acceptable salt,or hydrate thereof, and at least one additional therapeutic agent.Examples of additional therapeutic agents include, but are not limitedto, those listed above.

The composition, shape, and type of dosage forms of the invention willtypically vary depending on their use. For example, a dosage form usedin the acute treatment of a disease or a related disease may containlarger amounts of one or more of the active ingredients it comprisesthan a dosage form used in the chronic treatment of the same disease.Similarly, a parenteral dosage form may contain smaller amounts of oneor more of the active ingredients it comprises than an oral dosage formused to treat the same disease or disorder. These and other ways inwhich specific dosage forms encompassed by this invention will vary fromone another will be readily apparent to those skilled in the art. See,e.g., Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing,Easton Pa. (1990). Examples of dosage forms include, but are not limitedto: tablets; caplets; capsules, such as soft elastic gelatin capsules;cachets; troches; lozenges; dispersions; suppositories; ointments;cataplasms (poultices); pastes; powders; dressings; creams; plasters;solutions; patches; aerosols (e.g., nasal sprays or inhalers); gels;liquid dosage forms suitable for oral or mucosal administration to apatient, including suspensions (e.g., aqueous or non-aqueous liquidsuspensions, oil-in-water emulsions, or a water-in-oil liquidemulsions), solutions, and elixirs; liquid dosage forms suitable forparenteral administration to a patient; and sterile solids (e.g.,crystalline or amorphous solids) that can be reconstituted to provideliquid dosage forms suitable for parenteral administration to a patient.

Typical pharmaceutical compositions and dosage forms comprise one ormore carriers, excipients or diluents. Suitable excipients are wellknown to those skilled in the art of pharmacy, and non-limiting examplesof suitable excipients are provided herein. Whether a particularexcipient is suitable for incorporation into a pharmaceuticalcomposition or dosage form depends on a variety of factors well known inthe art including, but not limited to, the way in which the dosage formwill be administered to a patient. For example, oral dosage forms suchas tablets may contain excipients not suited for use in parenteraldosage forms. The suitability of a particular excipient may also dependon the specific active ingredients in the dosage form.

This invention further encompasses anhydrous pharmaceutical compositionsand dosage forms comprising active ingredients, since water canfacilitate the degradation of some compounds. For example, the additionof water (e.g., 5%) is widely accepted in the pharmaceutical arts as ameans of simulating long-term storage in order to determinecharacteristics such as shelf-life or the stability of formulations overtime. See, e.g., Jens T. Carstensen, Drug Stability: Principles &Practice, 2d. Ed., Marcel Dekker, NY, N.Y., 1995, pp. 379-80. In effect,water and heat accelerate the decomposition of some compounds. Thus, theeffect of water on a formulation can be of great significance sincemoisture and/or humidity are commonly encountered during manufacture,handling, packaging, storage, shipment, and use of formulations.

Anhydrous pharmaceutical compositions and dosage forms of the inventioncan be prepared using anhydrous or low moisture containing ingredientsand low moisture or low humidity conditions.

An anhydrous pharmaceutical composition should be prepared and storedsuch that its anhydrous nature is maintained. Accordingly, anhydrouscompositions are preferably packaged using materials known to preventexposure to water such that they can be included in suitable formularykits. Examples of suitable packaging include, but are not limited to,hermetically sealed foils, plastics, unit dose containers (e.g., vials),blister packs, and strip packs.

The invention further encompasses pharmaceutical compositions and dosageforms that comprise one or more compounds that reduce the rate by whichan active ingredient will decompose. Such compounds, which are referredto herein as “stabilizers,” include, but are not limited to,antioxidants such as ascorbic acid, pH buffers, or salt buffers.

Like the amounts and types of excipients, the amounts and specific typesof active ingredients in a dosage form may differ depending on factorssuch as, but not limited to, the route by which it is to be administeredto patients. However, typical dosage forms of the invention comprisecompounds of the invention, or a pharmaceutically acceptable salt orhydrate thereof comprise 0.1 mg to 1500 mg per unit to provide doses ofabout 0.01 to 200 mg/kg per day.

Oral Dosage Forms

Pharmaceutical compositions of the invention that are suitable for oraladministration can be presented as discrete dosage forms, such as, butare not limited to, tablets (e.g., chewable tablets), caplets, capsules,and liquids (e.g., flavored syrups). Such dosage forms containpredetermined amounts of active ingredients, and may be prepared bymethods of pharmacy well known to those skilled in the art. Seegenerally, Remington's Pharmaceutical Sciences, 18th ed., MackPublishing, Easton Pa. (1990).

Typical oral dosage forms of the invention are prepared by combining theactive ingredient(s) in an intimate admixture with at least oneexcipient according to conventional pharmaceutical compoundingtechniques. Excipients can take a wide variety of forms depending on theform of preparation desired for administration. For example, excipientssuitable for use in oral liquid or aerosol dosage forms include, but arenot limited to, water, glycols, oils, alcohols, flavoring agents,preservatives, and coloring agents. Examples of excipients suitable foruse in solid oral dosage forms (e.g., powders, tablets, capsules, andcaplets) include, but are not limited to, starches, sugars,micro-crystalline cellulose, diluents, granulating agents, lubricants,binders, and disintegrating agents.

Because of their ease of administration, tablets and capsules representthe most advantageous oral dosage unit forms, in which case solidexcipients are employed. If desired, tablets can be coated by standardaqueous or nonaqueous techniques. Such dosage forms can be prepared byany of the methods of pharmacy. In general, pharmaceutical compositionsand dosage forms are prepared by uniformly and intimately admixing theactive ingredients with liquid carriers, finely divided solid carriers,or both, and then shaping the product into the desired presentation ifnecessary.

For example, a tablet can be prepared by compression or molding.Compressed tablets can be prepared by compressing in a suitable machinethe active ingredients in a free-flowing form such as powder orgranules, optionally mixed with an excipient. Molded tablets can be madeby molding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent. Examples of excipients that canbe used in oral dosage forms of the invention include, but are notlimited to, binders, fillers, disintegrants, and lubricants. Binderssuitable for use in pharmaceutical compositions and dosage formsinclude, but are not limited to, corn starch, potato starch, or otherstarches, gelatin, natural and synthetic gums such as acacia, sodiumalginate, alginic acid, other alginates, powdered tragacanth, guar gum,cellulose and its derivatives (e.g., ethyl cellulose, cellulose acetate,carboxymethyl cellulose calcium, sodium carboxymethyl cellulose),polyvinyl pyrrolidone, methyl cellulose, pre-gelatinized starch,hydroxypropyl methyl cellulose, (e.g., Nos. 2208, 2906, 2910),microcrystalline cellulose, and mixtures thereof.

Examples of fillers suitable for use in the pharmaceutical compositionsand dosage forms disclosed herein include, but are not limited to, talc,calcium carbonate (e.g., granules or powder), microcrystallinecellulose, powdered cellulose, dextrates, kaolin, mannitol, silicicacid, sorbitol, starch, pre-gelatinized starch, and mixtures thereof.The binder or filler in pharmaceutical compositions of the invention istypically present in from about 50 to about 99 weight percent of thepharmaceutical composition or dosage form.

Suitable forms of microcrystalline cellulose include, but are notlimited to, the materials sold as AVICEL-PH-101, AVICEL-PH-103 AVICELRC-581, AVICEL-PH-105 (available from FMC Corporation, American ViscoseDivision, Avicel Sales, Marcus Hook, Pa.), and mixtures thereof. Anspecific binder is a mixture of microcrystalline cellulose and sodiumcarboxymethyl cellulose sold as AVICEL RC-581. Suitable anhydrous or lowmoisture excipients or additives include AVICEL-PH-103™ and Starch 1500LM.

Disintegrants are used in the compositions of the invention to providetablets that disintegrate when exposed to an aqueous environment.Tablets that contain too much disintegrant may disintegrate in storage,while those that contain too little may not disintegrate at a desiredrate or under the desired conditions. Thus, a sufficient amount ofdisintegrant that is neither too much nor too little to detrimentallyalter the release of the active ingredients should be used to form solidoral dosage forms of the invention. The amount of disintegrant usedvaries based upon the type of formulation, and is readily discernible tothose of ordinary skill in the art. Typical pharmaceutical compositionscomprise from about 0.5 to about 15 weight percent of disintegrant,specifically from about 1 to about 5 weight percent of disintegrant.

Disintegrants that can be used in pharmaceutical compositions and dosageforms of the invention include, but are not limited to, agar-agar,alginic acid, calcium carbonate, microcrystalline cellulose,croscarmellose sodium, crospovidone, polacrilin potassium, sodium starchglycolate, potato or tapioca starch, pre-gelatinized starch, otherstarches, clays, other algins, other celluloses, gums, and mixturesthereof.

Lubricants that can be used in pharmaceutical compositions and dosageforms of the invention include, but are not limited to, calciumstearate, magnesium stearate, mineral oil, light mineral oil, glycerin,sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid,sodium lauryl sulfate, talc, hydrogenated vegetable oil (e.g., peanutoil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, andsoybean oil), zinc stearate, ethyl oleate, ethyl laureate, agar, andmixtures thereof. Additional lubricants include, for example, a syloidsilica gel (AEROSIL 200, manufactured by W.R. Grace Co. of Baltimore,Md.), a coagulated aerosol of synthetic silica (marketed by Degussa Co.of Plano, Tex.), CAB-O-SIL (a pyrogenic silicon dioxide product sold byCabot Co. of Boston, Mass.), and mixtures thereof. If used at all,lubricants are typically used in an amount of less than about 1 weightpercent of the pharmaceutical compositions or dosage forms into whichthey are incorporated.

Delayed Release Dosage Forms

Active ingredients of the invention can be administered by controlledrelease means or by delivery devices that are well known to those ofordinary skill in the art. Examples include, but are not limited to,those described in U.S. Pat. Nos. 3,845,770; 3,916,899; 3,536,809;3,598,123; and 4,008,719, 5,674,533, 5,059,595, 5,591,767, 5,120,548,5,073,543, 5,639,476, 5,354,556, and 5,733,566, each of which isincorporated herein by reference. Such dosage forms can be used toprovide slow or controlled-release of one or more active ingredientsusing, for example, hydropropylmethyl cellulose, other polymer matrices,gels, permeable membranes, osmotic systems, multilayer coatings,microparticles, liposomes, microspheres, or a combination thereof toprovide the desired release profile in varying proportions. Suitablecontrolled-release formulations known to those of ordinary skill in theart, including those described herein, can be readily selected for usewith the active ingredients of the invention. The invention thusencompasses single unit dosage forms suitable for oral administrationsuch as, but not limited to, tablets, capsules, gelcaps, and capletsthat are adapted for controlled-release.

All controlled-release pharmaceutical products have a common goal ofimproving drug therapy over that achieved by their non-controlledcounterparts. Ideally, the use of an optimally designedcontrolled-release preparation in medical treatment is characterized bya minimum of drug substance being employed to cure or control thecondition in a minimum amount of time. Advantages of controlled-releaseformulations include extended activity of the drug, reduced dosagefrequency, and increased patient compliance. In addition,controlled-release formulations can be used to affect the time of onsetof action or other characteristics, such as blood levels of the drug,and can thus affect the occurrence of side (e.g., adverse) effects.

Most controlled-release formulations are designed to initially releasean amount of drug (active ingredient) that promptly produces the desiredtherapeutic effect, and gradually and continually release of otheramounts of drug to maintain this level of therapeutic or prophylacticeffect over an extended period of time. In order to maintain thisconstant level of drug in the body, the drug must be released from thedosage form at a rate that will replace the amount of drug beingmetabolized and excreted from the body. Controlled-release of an activeingredient can be stimulated by various conditions including, but notlimited to, pH, temperature, enzymes, water, or other physiologicalconditions or compounds.

Parenteral Dosage Forms

Parenteral dosage forms can be administered to patients by variousroutes including, but not limited to, subcutaneous, intravenous(including bolus injection), intramuscular, and intraarterial. Becausetheir administration typically bypasses patients' natural defensesagainst contaminants, parenteral dosage forms are preferably sterile orcapable of being sterilized prior to administration to a patient.Examples of parenteral dosage forms include, but are not limited to,solutions ready for injection, dry and/or lyophylized products ready tobe dissolved or suspended in a pharmaceutically acceptable vehicle forinjection (reconstitutable powders), suspensions ready for injection,and emulsions.

Suitable vehicles that can be used to provide parenteral dosage forms ofthe invention are well known to those skilled in the art. Examplesinclude, but are not limited to: Water for Injection USP; aqueousvehicles such as, but not limited to, Sodium Chloride Injection,Ringer's Injection, Dextrose Injection, Dextrose and Sodium ChlorideInjection, and Lactated Ringer's Injection; water-miscible vehicles suchas, but not limited to, ethyl alcohol, polyethylene glycol, andpolypropylene glycol; and non-aqueous vehicles such as, but not limitedto, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate,isopropyl myristate, and benzyl benzoate. Compounds that increase thesolubility of one or more of the active ingredients disclosed herein canalso be incorporated into the parenteral dosage forms of the invention.

Transdermal Dosage Forms

Transdermal dosage forms include “reservoir type” or “matrix type”patches, which can be applied to the skin and worn for a specific periodof time to permit the penetration of a desired amount of activeingredients.

Suitable excipients (e.g., carriers and diluents) and other materialsthat can be used to provide transdermal and topical dosage formsencompassed by this invention are well known to those skilled in thepharmaceutical arts, and depend on the particular tissue to which agiven pharmaceutical composition or dosage form will be applied. Withthat fact in mind, typical excipients include, but are not limited to,water, acetone, ethanol, ethylene glycol, propylene glycol,butane-1,3-diol, isopropyl myristate, isopropyl palmitate, mineral oil,and mixtures thereof. Depending on the specific tissue to be treated,additional components may be used prior to, in conjunction with, orsubsequent to treatment with active ingredients of the invention. Forexample, penetration enhancers can be used to assist in delivering theactive ingredients to the tissue. Suitable penetration enhancersinclude, but are not limited to: acetone; various alcohols such asethanol, oleyl, and tetrahydrofuryl; alkyl sulfoxides such as dimethylsulfoxide; dimethyl acetamide; dimethyl formamide; polyethylene glycol;pyrrolidones such as polyvinylpyrrolidone; Kollidon grades (Povidone,Polyvidone); urea; and various water-soluble or insoluble sugar esterssuch as Tween 80 (polysorbate 80) and Span 60 (sorbitan monostearate).

The pH of a pharmaceutical composition or dosage form, or of the tissueto which the pharmaceutical composition or dosage form is applied, mayalso be adjusted to improve delivery of one or more active ingredients.Similarly, the polarity of a solvent carrier, its ionic strength, ortonicity can be adjusted to improve delivery. Compounds such asstearates can also be added to pharmaceutical compositions or dosageforms to advantageously alter the hydrophilicity or lipophilicity of oneor more active ingredients so as to improve delivery. In this regard,stearates can serve as a lipid vehicle for the formulation, as anemulsifying agent or surfactant, and as a delivery-enhancing orpenetration-enhancing agent. Different salts, hydrates or solvates ofthe active ingredients can be used to further adjust the properties ofthe resulting composition.

Topical Dosage Forms

Topical dosage forms of the invention include, but are not limited to,creams, lotions, ointments, gels, solutions, emulsions, suspensions, orother forms known to one of skill in the art. See, e.g., Remington'sPharmaceutical Sciences, 18th eds., Mack Publishing, Easton Pa. (1990);and Introduction to Pharmaceutical Dosage Forms, 4th ed., Lea & Febiger,Philadelphia (1985).

Suitable excipients (e.g., carriers and diluents) and other materialsthat can be used to provide transdermal and topical dosage formsencompassed by this invention are well known to those skilled in thepharmaceutical arts, and depend on the particular tissue to which agiven pharmaceutical composition or dosage form will be applied. Withthat fact in mind, typical excipients include, but are not limited to,water, acetone, ethanol, ethylene glycol, propylene glycol,butane-1,3-diol, isopropyl myristate, isopropyl palmitate, mineral oil,and mixtures thereof.

Depending on the specific tissue to be treated, additional componentsmay be used prior to, in conjunction with, or subsequent to treatmentwith active ingredients of the invention. For example, penetrationenhancers can be used to assist in delivering the active ingredients tothe tissue. Suitable penetration enhancers include, but are not limitedto: acetone; various alcohols such as ethanol, oleyl, andtetrahydrofuryl; alkyl sulfoxides such as dimethyl sulfoxide; dimethylacetamide; dimethyl formamide; polyethylene glycol; pyrrolidones such aspolyvinylpyrrolidone; Kollidon grades (Povidone, Polyvidone); urea; andvarious water-soluble or insoluble sugar esters such as Tween 80(polysorbate 80) and Span 60 (sorbitan monostearate).

Mucosal Dosage Forms

Mucosal dosage forms of the invention include, but are not limited to,ophthalmic solutions, sprays and aerosols, or other forms known to oneof skill in the art. See, e.g., Remington's Pharmaceutical Sciences,18th eds., Mack Publishing, Easton Pa. (1990); and Introduction toPharmaceutical Dosage Forms, 4th ed., Lea & Febiger, Philadelphia(1985). Dosage forms suitable for treating mucosal tissues within theoral cavity can be formulated as mouthwashes or as oral gels. In oneembodiment, the aerosol comprises a carrier. In another embodiment, theaerosol is carrier free.

The compounds of the invention may also be administered directly to thelung by inhalation. For administration by inhalation, a compound of theinventionr can be conveniently delivered to the lung by a number ofdifferent devices. For example, a Metered Dose Inhaler (“MDI”) whichutilizes canisters that contain a suitable low boiling propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas can beused to deliver a compound directly to the lung. MDI devices areavailable from a number of suppliers such as 3M Corporation, Aventis,Boehringer Ingleheim, Forest Laboratories, Glaxo-Wellcome, ScheringPlough and Vectura.

Alternatively, a Dry Powder Inhaler (DPI) device can be used toadminister a compound of the invention to the lung (see, e.g., Raleighet al., Proc. Amer. Assoc. Cancer Research Annual Meeting, 1999, 40,397, which is herein incorporated by reference). DPI devices typicallyuse a mechanism such as a burst of gas to create a cloud of dry powderinside a container, which can then be inhaled by the patient. DPIdevices are also well known in the art and can be purchased from anumber of vendors which include, for example, Fisons, Glaxo-Wellcome,Inhale Therapeutic Systems, ML Laboratories, Qdose and Vectura. Apopular variation is the multiple dose DPI (“MDDPI”) system, whichallows for the delivery of more than one therapeutic dose. MDDPI devicesare available from companies such as AstraZeneca, GlaxoWellcome, WAX,Schering Plough, SkyePharma and Vectura. For example, capsules andcartridges of gelatin for use in an inhaler or insufflator can beformulated containing a powder mix of the compound and a suitable powderbase such as lactose or starch for these systems.

Another type of device that can be used to deliver a compound of theinvention to the lung is a liquid spray device supplied, for example, byAradigm Corporation. Liquid spray systems use extremely small nozzleholes to aerosolize liquid drug formulations that can then be directlyinhaled into the lung.

In a preferred embodiment, a nebulizer device is used to deliver acompound of the invention to the lung. Nebulizers create aerosols fromliquid drug formulations by using, for example, ultrasonic energy toform fine particles that can be readily inhaled (See e.g., Verschoyle etal., British J. Cancer, 1999, 80, Suppl 2, 96, which is hereinincorporated by reference). Examples of nebulizers include devicessupplied by Sheffield/Systemic Pulmonary Delivery Ltd. (See, Armer etal., U.S. Pat. No. 5,954,047; van der Linden et al., U.S. Pat. No.5,950,619; van der Linden et al., U.S. Pat. No. 5,970,974, which areherein incorporated by reference), Aventis and Batelle PulmonaryTherapeutics.

In a particularly preferred embodiment, an electrohydrodynamic (“EHD”)aerosol device is used to deliver compounds of the invention to thelung. EHD aerosol devices use electrical energy to aerosolize liquiddrug solutions or suspensions (see, e.g., Noakes et al., U.S. Pat. No.4,765,539; Coffee, U.S. Pat. No. 4,962,885; Coffee, PCT Application, WO94/12285; Coffee, PCT Application, WO 94/14543; Coffee, PCT Application,WO 95/26234, Coffee, PCT Application, WO 95/26235, Coffee, PCTApplication, WO 95/32807, which are herein incorporated by reference).The electrochemical properties of the formulation may be importantparameters to optimize when delivering this drug to the lung with an EHDaerosol device and such optimization is routinely performed by one ofskill in the art. EHD aerosol devices may more efficiently deliverydrugs to the lung than existing pulmonary delivery technologies. Othermethods of intra-pulmonary delivery of the compounds of the inventionwill be known to the skilled artisan and are within the scope of theinvention.

Liquid drug formulations suitable for use with nebulizers and liquidspray devices and EHD aerosol devices will typically include a compoundof the invention with a pharmaceutically acceptable carrier. Preferably,the pharmaceutically acceptable carrier is a liquid such as alcohol,water, polyethylene glycol or a perfluorocarbon. Optionally, anothermaterial may be added to alter the aerosol properties of the solution orsuspension of the compound. Preferably, this material is liquid such asan alcohol, glycol, polyglycol or a fatty acid. Other methods offormulating liquid drug solutions or suspension suitable for use inaerosol devices are known to those of skill in the art (see, e.g.,Biesalski, U.S. Pat. Nos. 5,112,598; Biesalski, 5,556,611, which areherein incorporated by reference) A compound can also be formulated inrectal or vaginal compositions such as suppositories or retentionenemas, e.g., containing conventional suppository bases such as cocoabutter or other glycerides.

In addition to the formulations described previously, a compound of theinvention can also be formulated as a depot preparation. Such longacting formulations can be administered by implantation (for examplesubcutaneously or intramuscularly) or by intramuscular injection. Thus,for example, the compounds can be formulated with suitable polymeric orhydrophobic materials (for example, as an emulsion in an acceptable oil)or ion exchange resins, or as sparingly soluble derivatives, forexample, as a sparingly soluble salt.

Alternatively, other pharmaceutical delivery systems can be employed.Liposomes and emulsions are well known examples of delivery vehiclesthat can be used to deliver the compounds of the invention. Certainorganic solvents such as dimethylsulfoxide can also be employed,although usually at the cost of greater toxicity. The compounds of theinvention can also be delivered in a controlled release system. In oneembodiment, a pump can be used (Sefton, CRC Crit. Ref Biomed Eng., 1987,14, 201; Buchwald et al., Surgery, 1980, 88, 507; Saudek et al., N.Engl. J. Med., 1989, 321, 574). In another embodiment, polymericmaterials can be used (see Medical Applications of Controlled Release,Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); ControlledDrug Bioavailability, Drug Product Design and Performance, Smolen andBall (eds.), Wiley, New York (1984); Ranger and Peppas, J. Macromol.Sci. Rev. Macromol. Chem., 1983, 23, 61; see also Levy et al., Science,1985, 228, 190; During et al., Ann. Neurol., 1989, 25, 351; Howard etal., 1989, J. Neurosurg. 71, 105). In yet another embodiment, acontrolled-release system can be placed in proximity of the target ofthe compounds of the invention, e.g., the lung, thus requiring only afraction of the systemic dose (see, e.g., Goodson, in MedicalApplications of Controlled Release, supra, vol. 2, pp. 115 (1984)).Other controlled-release system can be used (see, e.g. Langer, Science,1990, 249, 1527).

Suitable excipients (e.g., carriers and diluents) and other materialsthat can be used to provide mucosal dosage forms encompassed by thisinvention are well known to those skilled in the pharmaceutical arts,and depend on the particular site or method which a given pharmaceuticalcomposition or dosage form will be administered. With that fact in mind,typical excipients include, but are not limited to, water, ethanol,ethylene glycol, propylene glycol, butane-1,3-diol, isopropyl myristate,isopropyl palmitate, mineral oil, and mixtures thereof, which arenon-toxic and pharmaceutically acceptable. Examples of such additionalingredients are well known in the art. See, e.g., Remington'sPharmaceutical Sciences, 18th eds., Mack Publishing, Easton Pa. (1990).

The pH of a pharmaceutical composition or dosage form, or of the tissueto which the pharmaceutical composition or dosage form is applied, canalso be adjusted to improve delivery of one or more active ingredients.Similarly, the polarity of a solvent carrier, its ionic strength, ortonicity can be adjusted to improve delivery. Compounds such asstearates can also be added to pharmaceutical compositions or dosageforms to advantageously alter the hydrophilicity or lipophilicity of oneor more active ingredients so as to improve delivery. In this regard,stearates can serve as a lipid vehicle for the formulation, as anemulsifying agent or surfactant, and as a delivery-enhancing orpenetration-enhancing agent. Different salts, hydrates or solvates ofthe active ingredients can be used to further adjust the properties ofthe resulting composition.

Kits

The invention provides a pharmaceutical pack or kit comprising one ormore containers comprising a Formula I prodrug, Formula II compound, orother compound of the invention useful for the treatment or preventionof a Hepatitis C virus infection. In other embodiments, the inventionprovides a pharmaceutical pack or kit comprising one or more containerscomprising a compound of the invention useful for the treatment orprevention of a Hepatitis C virus infection and one or more containerscomprising an additional therapeutic agent, including but not limited tothose listed above, in particular an antiviral agent, an interferon, anagent which inhibits viral enzymes, or an agent which inhibits viralreplication, preferably the additional therapeutic agent is HCV specificor demonstrates anti-HCV activity.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers comprising one or more of the ingredients of thepharmaceutical compositions of the invention. Optionally associated withsuch container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration.

The inventive agents may be prepared using the reaction routes andsynthesis schemes as described below, employing the general techniquesknown in the art using starting materials that are readily available.The synthesis of non-exemplified compounds according to the inventionmay be successfully performed by modifications apparent to those skilledin the art, e.g., by appropriately protecting interfering groups, bychanging to other suitable reagents known in the art, or by makingroutine modifications of reaction conditions. Alternatively, otherreactions disclosed herein or generally known in the art will berecognized as having applicability for preparing other compounds of theinvention.

Preparation of Compounds

In the synthetic schemes described below, unless otherwise indicated alltemperatures are set forth in degrees Celsius and all parts andpercentages are by weight. Reagents were purchased from commercialsuppliers such as Aldrich Chemical Company or Lancaster Synthesis Ltd.and were used without further purification unless otherwise indicated.Tetrahydrofuran (THF) and N,N-dimethylforamide (DMF) were purchased fromAldrich in Sure Seal bottles and used as received. Unless otherwiseindicated, the following solvents and reagents were distilled under ablanket of dry nitrogen. THF, and Et₂O were distilled fromNa-benzophenone ketyl; CH₂Cl₂, diisopropylamine, pyridine and Et₃N weredistilled from CaH₂; MeCN was distilled first from P₂O₅, then from CaH₂;MeOH was distilled from Mg; PhMe, EtOAc and i-PrOAc were distilled fromCaH₂; TFAA was purified via simple atmospheric distillation under dryargon.

The reactions set forth below were done generally under a positivepressure of argon at an ambient temperature (unless otherwise stated) inanhydrous solvents, and the reaction flasks were fitted with rubbersepta for the introduction of substrates and reagents via syringe.Glassware was oven dried and/or heat dried. The reactions were assayedby TLC and terminated as judged by the consumption of starting material.Analytical thin layer chromatography (TLC) was performed onaluminum-backed silica gel 60 F₂₅₄ 0.2 mm plates (EM Science), andvisualized with UV light (254 nm) followed by heating with commercialethanolic phosphomolybdic acid. Preparative thin layer chromatography(TLC) was performed on aluminum-backed silica gel 60 F₂₅₄ 1.0 mm plates(EM Science) and visualized with UV light (254 nm).

Work-ups were typically done by doubling the reaction volume with thereaction solvent or extraction solvent and then washing with theindicated aqueous solutions using 25% by volume of the extraction volumeunless otherwise indicated. Product solutions were dried over anhydrousNa₂SO₄ and/or Mg₂SO₄ prior to filtration and evaporation of the solventsunder reduced pressure on a rotary evaporator and noted as solventsremoved in vacuo. Column chromatography was completed under positivepressure using 230-400 mesh silica gel or 50-200 mesh neutral alumina.Hydrogenolysis was done at the pressure indicated in the examples or atambient pressure.

¹H-NMR spectra were recorded on a Varian Mercury-VX400 instrumentoperating at 400 MHz and ¹³C-NMR spectra were recorded operating at 75MHz. NMR spectra were obtained as CDCl₃ solutions (reported in ppm),using chloroform as the reference standard (7.27 ppm and 77.00 ppm),CD₃OD (3.4 and 4.8 ppm and 49.3 ppm), DMSO-d₆, or internallytetramethylsilane (0.00 ppm) when appropriate. Other NMR solvents wereused as needed. When peak multiplicities are reported, the followingabbreviations are used: s (singlet), d (doublet), t (triplet), q(quartet), m (multiples), br (broadened), dd (doublet of doublets), dt(doublet of triplets). Coupling constants, when given, are reported inHertz (Hz).

Infrared (IR) spectra were recorded on a FT-IR Spectrometer as neatoils, as KBr pellets, or as CDCl₃ solutions, and when given are reportedin wave numbers (cm⁻¹). Mass spectra reported are (+)-ES LC/MS conductedby the Analytical Chemistry Department of Anadys Pharmaceuticals, Inc.Elemental analyses were conducted by the Atlantic Microlab, Inc. inNorcross, Ga. Melting points (mp) were determined on an open capillaryapparatus, and are uncorrected.

The described synthetic pathways and experimental procedures utilizemany common chemical abbreviations, THF (tetrahydrofuran), DMF(N,N-dimethylformamide), EtOAc (ethyl acetate), DMSO (di-methylsulfoxide), DMAP (4-dimethylaminopyridine), DBU(1,8-diazacyclo[5.4.0]undec-7-ene), DCM(4-(dicyanomethylene)-2-methyl-6-(4-dimethylamino-styryl)-4H-pyran),MCPBA (3-chloroperoxybenzoic acid), EDC(1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride), HATU(O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate), HOBT (1-hydroxybenzotriazole hydrate), TFAA(trifluoroacetic anhydride), pyBOP(benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate),DIEA (diisopropylethylamine), BOC (tert-butoxycarbonyl), 2,2-DMP(2,2-dimethoxypropane), IPA (isopropyl alcohol), TEA (triethylamine),DCE (1,2-dichloroethane), PPTS (pyridinium p-toluenesulfonate), DEAD(diethylazodicarboxylate), PS (polymer supported), HF (hydrogenfluoride), MeCN (acetonitrile), MeOH (methanol), Val (valine), Phe(phenyl alanine), HPLC (high pressure liquid chromatography), TLC (thinlayer chromatography), Bz (benzoyl), Ac (acetyl), Tol (toluoyl), Me(methyl), and the like.

Example 15-Amino-3-(2′-C-methyl-β-D-ribofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one(4)

Step 1) Preparation of5-Amino-3-(2′-C-methyl-2′,3′,5′-tri-O-benzoyl-β-D-ribofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one(3)

To a heterogeneous mixture of heterocycle 1 (168 mg, 1.00 mmol) andperbenzoyl ribose 2 [prepared according to the method of Wolfe et al. J.Org. Chem. 1997, 62, 1754-1759] (522 mg, 0.90 mmol) in anhydrous MeCN(10 mL) was added BSA (742 μL, 3.00 mmol). The resultant mixture wasstirred 15 min whereupon TMSOTf (333 μL, 1.50 mmol) was added. Thereaction mixture was stirred for 4 h at 65° C., then 3 h at 90° C. Themixture was then cooled to rt, diluted with DCM (150 mL) and partitionedwith pH 7 buffer (100 mL). The aqueous phase was further extracted withDCM (3×50 mL), and the combined organic phases were dried over Na₂SO₄and filtered. The clear filtrate was diluted with EtOAc (200 mL),filtered through a short pad of SiO₂, concentrated and submitted toflash chromatography (10-40% EtOAc-DCM), affording 380 mg (67%) ofnucleoside 3 as a white solid: ¹H (400 MHz, DMSO-d₆) δ 8.43 (s, 1H),7.85-8.02 (m, 6H), 7.46-7.97 (m, 7H), 7.35 (t, J=8.06, 2H), 6.96 (br s,2H), 6.77 (br s, 1H), 4.54-4.82 (m, 4H), 1.77 (s, 3H); [M+H]⁺ m/z 627.

Step 2) Preparation of5-Amino-3-(2′-C-methyl-β-D-ribofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one(4)

To a suspension of nucleoside 3 (380 mg, 0.606 mmol) in MeOH (20 mL) wasadded K₂CO₃ (17 mg, 0.12 mmol) at rt. The resulting mixture was stirred18 h at rt whereupon it was treated with HOAc (15 μL, 0.25 mmol),concentrated and submitted to HPLC purification (MeCN—H₂O), affording 20mg (10%) of the title compound 4 as a white solid after lyophilization:¹H (400 MHz, DMSO-d₆) δ 8.33 (s, 1H), 6.86 (br s, 2H), 6.09 (br s, 1H),5.19 (br s, 1H), 4.88 (br s, 1H), 4.55 (t, J=5.87, 1H), 3.97 (br s, 1H),3.76-3.81 (m, 1H), 3.61 (br s, 2H), 1.04 (s, 3H); [M+H]⁺ m/z 315.Analysis calc'd for C₁₁H₁₄N₄O₅S.H₂O: C, 39.75; H, 4.85; N, 16.86; S,9.65. Found: C, 40.23; H, 4.76; N, 16.64; S, 9.45.

Example 25-Amino-3-(2′-deoxy-β-D-ribofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one(10)

Step 1) Preparation ofN′-(7-Chloro-2-oxo-2,3-dihydro-thiazolo[4,5-d]pyrimidin-5-yl)-N,N-dimethyl-formamidine(7)

To a suspension of 5-amino-7-hydroxy-3H-thiazolo[4,5-d]pyrimidin-2-one(10.0 g, 53.7 mmol) in MeCN (200 mL) at 0° C. was added SOCl₂ (20.0 mL,274 mmol) dropwise via addition funnel over 20 min. The resultingmixture was slowly warmed to rt then immersed into a 60° C. oil bathwhere it was stirred for 48 h. The reaction mixture was cooled to rt andslowly poured into 300 g of cracked ice in 300 mL of water containingNaHCO₃ (46 g, 548 mmol). The aqueous mixture was extracted with 20%IPA-DCM (3×500 mL), and the combined organic phases were dried overNa₂SO₄ and concentrated to a residue that was triturated with EtOAc toafford 6.33 g (46%) of chloroamidine 7 as a tan solid: ¹H (400 MHz,DMSO-d₆) δ 12.60 (s, 1H), 8.69 (s, 1H), 3.25 (s, 3H), 3.11 (s, 3H);[M+H]⁺ m/z 258.

Step 2) Preparation ofN′-(7-Chloro-2-oxo-3-[2′-deoxy-3′,5′-di-O-(p-toluoyl)-β-D-ribofuranosyl]-2,3-dihydro-thiazolo[4,5-d]pyrimidin-5-yl)-N,N-dimethyl-formamidine(8)

To a suspension of heterocycle 7 (1.79 g, 6.94 mmol) in anhydrous MeCN(90 mL) at rt was added 95% NaH (183 mg, 7.63 mmol). The resultingmixture was stirred 30 min whereupon chlorosugar 5 (2.70 g, 6.94 mmol)[purchased from Berry & Associates, Inc., Dexter, Mich.] was added. Thereaction mixture was heated to 55° C., stirred 1 h, cooled,concentrated, and then submitted to flash chromatography (SiO₂, 5-10%EtOAc-DCM) affording 3.8 g (90%) of nucleoside 8 as a solid materialthat may be further purified via trituration in MeOH: ¹H (400 MHz,DMSO-d₆) δ 8.64 (s, 1H), 7.83 (ABq, J_(AB)=8.19, Δv_(AB)=38.53, 4H),7.28 (ABq, J_(AB)=8.19, Δv_(AB)=36.13, 4H), 6.56 (dd, J=8.19, 5.07, 1H),5.76-5.80 (m, 1H), 4.56-4.60 (m, 1H), 4.45-4.50 (m, 2H), 3.27-3.34 (m,1H), 3.15 (s, 3H), 3.03 (s, 3H), 2.57-2.64 (m, 1H), 2.35 (s, 3H), 2.39(s, 3H).

Step 3) Preparation of5-Amino-3-(2′,3′-di-O-(p-toluoyl)-β-D-ribofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one(9)

To a solution of chloroaryl nucleoside 8 (924 mg, 1.47 mmol) in aceticacid (10.4 mL) at rt was added Zn—Cu couple (1.54 g, 11.9 mmol). Theresulting suspension was stirred vigorously at ambient temperature for3.5 h, filtered through celite, and then concentrated to a solidmaterial that was submitted to flash chromatography (SiO₂, 0-10%EtOAc-CHCl₃), yielding 520 mg (67%) of compound 9 as a tan solid: ¹H NMR(400 MHz, d₆-DMSO) δ 8.34 (s, 1H), 7.85 (ABq, J_(AB)=8.4, Δv_(AB)=17.6,4H), 7.30 (ABq, J_(AB)=8.4, Δv_(AB)=27.1, 4H), 6.87 (s, 2H), 6.47 (m,1H), 5.78 (m, 1H), 4.62 (m, 1H), 4.47 (m, 2H), 3.35 (m, 2H), 2.38 (s,3H), 2.35 (s, 3H).

Step 4) Preparation of5-Amino-3-(2′-deoxy-β-D-ribofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one(10)

To a suspension of diester 9 (300 mg, 0.577 mmol) from Step 3 (above) inMeOH (20 mL) at rt was added K₂CO₃ (188 mg, 1.36 mmol). The resultingmixture was stirred 8 h whereupon it was quenched with HOAc (164 μL,2.86 mmol), then concentrated and submitted to HPLC (MeCN—H₂O, TFA) toafford 75 mg (46%) of the title compound 10 as a white solid (TFA salt)after lyophilization: ¹H (400 MHz, DMSO-d₆) δ 8.34 (s, 1H), 7.15 (br s,2H), 6.33 (t, J=7.0, 1H), 4.33 (br s, 1H), 3.72-3.73 (m, 1H), 3.39-3.56(m, 2H), 2.89-2.96 (m, 1H), 1.99-2.05 (m, 1H); [M+H]⁺ m/z 285. Analysiscalc'd for C₁₀H₁₂N₄O₄S.C₂HF₃O₂: C, 36.18; H, 3.29; N, 14.31; S, 8.05.Found: C, 36.28; H, 3.35; N, 13.96; S, 8.05.

Example 35-Amino-3-(2′-O-acetyl-3′-deoxy-β-D-ribofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one(15)

Step 1) Preparation of5-Amino-3-(2′,5′-di-O-benzoyl-3′-deoxy-β-D-ribofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one(12)

To a heterogeneous mixture of heterocycle 1 (25.0 g, 0.149 mol) anddeoxyribofuranose 11a (47.0 g, 0.122 mol) [may be prepared from thecorresponding methyl ribofuranoside (Walton, et. al. J. Med. Chem. 1965,8, 659-663) via the method of Valdivia, et. al. Tetrahedron Lett. 2005,46, 6511-6514] in anhydrous MeCN (640 mL) at RT was added dropwise viaaddition funnel BSA (113 mL, 0.458 mol) over 20 min. The resultantsuspension was treated dropwise with TMSOTf (41.5 mL, 0.229 mol) at rtover 20 min, whereupon it became nearly homogeneous. The mixture washeated to reflux (internal T 83° C.) and stirred for 8 h, then cooled tort and concentrated to an oily residue via rotary evaporation. Theresidue was dissolved in EtOAc (500 mL) and cooled to 10° C. where itwas slowly treated with 1 M pH 7 phosphate buffer (400 mL), keeping theinternal temperature below 35° C. Upon completed addition of buffer, thepH of the mixture was adjusted to 7.0 with solid K₂HPO₄ and vigorousstirring was continued for 1 h. Celite (25 g) was added, and the mixturestirred an additional 30 min. Filtration of the triphasic mixturethrough a short pad of celite provided two clear phases. The aqueousphase was saturated with solid NaCl then extracted with EtOAc (4×250mL). The combined organic phases were washed with brine (400 mL), driedover Na₂SO₄ and charcoal (1 g), and then filtered through a short pad ofSiO₂. The clear amber filtrate was concentrated to dryness, whereuponsolid heterocycle had precipitated. The residue was taken up in DCM,treated with a small amount of MgSO₄, and then filtered through celite.The clear filtrate was concentrated and further dried under high vacuumat 35° C. to provide a tan, crispy foam (69.5 g). Submission of thissolid foam to flash chromatography (SiO₂, 5-40% EtOAc-hexanes) afforded46.3 g (77%) of nucleoside 12 as a light beige solid foam: ¹H NMR(DMSO-d₆) δ 8.35 (s, 1H), 7.93-8.01 (m, 4H), 7.61-7.69 (m, 2H),7.47-7.56 (m, 4H), 6.94 (s, 2H), 6.09 (d, J=1.9, 1H), 6.00 (d, J=7.4,1H), 4.64-4.69 (m, 1H), 4.57 (dd, J=12.1, 2.7, 1H), 4.36 (dd, J=12.1,5.8, 1H), 2.92-3.00 (m, 1H), 2.32 (dd, J=14.0, 5.8, 1H); [M+H]⁺ m/z 493.

Step 2) Preparation of5-Amino-3-(3′-deoxy-β-D-ribofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one(13)

To a heterogeneous mixture of dibenzoate 12 (46.3 g, 94.0 mmol) andanhydrous MeOH (1.0 L) was added K₂CO₃ (2.59 g, 18.8 mmol) at rt. Themixture became homogeneous within 30 min, then heterogeneous againwithin 3 h. Additional MeOH (100 mL) was added to increase fluidity, andthe reaction mixture was stirred for a total of 24 h. The suspension wastreated with HOAc (2.26 mL, 39.5 mmol) and then concentrated at 45° C.whereupon it was cooled, then triturated with EtOH (200 mL) and ether(1800 mL) for 1 h. The solid material was filtered, washed with ether(3×250 mL), air dried and then washed with water (2×250 mL), affording19.47 g (78%) of diol 13 as a white solid that was dried in vaccuo andrecrystallized from water: ¹H NMR (DMSO-d₆) δ 8.31 (s, 1H), 6.82 (s,2H), 5.82 (d, 1H), 5.41 (d, 1H), 4.79-4.83 (m, 1H), 4.65 (t, J=5.8, 1H),4.13-4.20 (m, 1H), 3.40-3.49 (m, 2H), 2.31 (ddd, J=16.0, 9.4, 7.0, 1H),1.81 (ddd, J=12.5, 5.8, 2.3, 1H); [M+H]⁺ m/z 285. Analysis calc'd forC₁₀H₁₂N₄O₄S: C, 42.25; H, 4.25; N, 19.71; S, 11.28. Found: C, 42.36; H,4.32; N, 19.72; S, 11.23.

Step 3) Preparation of5-Amino-3-(2′,5′-di-O-acetyl-3′-deoxy-β-D-ribofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one(14)

To a suspension of diol 13 (8.00 g, 28.1 mmol), Et₃N (11.8 mL, 84.4mmol), and DMAP (344 mg, 2.81 mmol) in anhydrous MeCN (190 mL) at 0° C.was added dropwise Ac₂O (5.44 mL, 57.7 mmol). The resultant mixture,that became homogeneous within 1.5 h, was slowly warmed to rt andstirred for 18 h whereupon it was concentrated to a residue that wassubmitted to flash chromatography (SiO₂, 0-100% EtOAc-DCM) to afford8.34 g (80%) of diacetate 14 as a white solid foam that may be furtherpurified via trituration with ether-hexanes: ¹H (400 MHz, DMSO-d₆) δ8.34 (s, 1H), 6.91 (s, 2H), 5.90 (d, J=1.9, 1H), 5.65 (d, J=7.4, 1H),4.33-4.39 (m, 1H), 4.25 (dd, J=12.1, 3.1, 1H), 4.01 (dd, J=11.7, 6.6,1H), 2.65-2.73 (m, 1H), 2.06 (dd, J=13.6, 6.2, 1H), 2.05 (s, 3H), 1.98(s, 3H); [M+H]⁺ m/z 369. Analysis calc'd for C₁₄H₁₆N₄O₆S: C, 45.65; H,4.38; N, 15.21; S, 8.70. Found: C, 45.69; H, 4.52; N, 15.02; S, 8.64.

Alternatively diacetate 14 may be prepared from heterocycle 1 anddeoxyribo-furanose 11b [may be prepared via the method of Valdivia, et.al. Tetrahedron Lett. 2005, 46, 659-663] in a manner similar to Step 1above with a yield of 63%.

Step 4) Preparation of5-Amino-3-(2′-O-acetyl-3′-deoxy-β-D-ribofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one(15)

To a slowly stirring suspension of diacetate 14 (5.08 g, 13.8 mmol) andCandida Arctica lipase acrylic resin (2.50 g) [purchased from Sigma] inacetone (50 mL) was added 50 mM pH 7 phosphate buffer (250 mL) at rt.The resulting mixture was stirred slowly for 18 h whereupon it wasfiltered, concentrated and extracted with EtOAc (4×250 mL). The combinedorganic phases were dried over Na₂SO₄, concentrated and then submittedto flash chromatography (SiO₂, 0-15% IPA-DCM) to afford 4.11 g (91%) ofthe title compound 15 as a white solid that may be further purified viatrituration with ether-hexanes: ¹H (400 MHz, DMSO-d₆) δ 8.33 (s, 1H),6.87 (s, 2H), 5.88 (d, J=2.3, 1H), 5.66 (d, J=7.8, 1H), 4.76 (t, J=5.8,1H), 4.11-4.18 (m, 1H), 3.43-3.53 (m, 2H), 2.50-2.57 (m, 1H), 2.05 (s,3H), 1.98 (dd, J=13.6, 5.8, 1H); [M+H]⁺ m/z 327. Analysis calc'd forC₁₂H₁₄N₄O₅S: C, 44.17; H, 4.32; N, 17.17; S, 9.83. Found: C, 44.12; H,4.45; N, 16.88; S, 9.69.

Example 45-Amino-3-(2′-O-acetyl-5′-O-benzoyl-3′-deoxy-β-D-ribofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one(16)

To a heterogeneous mixture of heterocycle 1 (106 mg, 0.633 mmol) anddeoxyribofuranose 11c [purchased from Berry & Associates, Inc., Dexter,Mich.] (183 mg, 0.57 mmol) in anhydrous MeCN (8 mL) was added BSA (464uL, 1.89 mmol) at rt. The resultant mixture was immersed into a 60° C.oil bath and stirred for 4 h. The mixture was concentrated via rotaryevaporation and partitioned between EtOAc (100 mL) and saturated NaHCO₃(50 mL). The organic phase was dried over Na₂SO₄ and then concentrated.The crude material was triturated with Et₂O-EtOAc to afford 132 mg (54%)of an off-white solid that was submitted to HPLC (MeCN—H₂O) to providean analytical sample: ¹H (400 MHz, DMSO-d₆) δ 8.33 (s, 1H), 7.91-7.94(m, 2H), 7.60-7.65 (m, 1H), 7.47-7.50 (m, 2H), 6.94 (s, 2H), 5.93 (d,J=1.8, 1H), 5.72 (dd, J=5.9, 1.5, 1H), 4.50-4.53 (m, 2H), 4.31 (q,J=7.0, 1H), 2.80-2.88 (m, 1H), 2.14 (dd, J=13.2, 5.1, 1H), 2.07 (s, 3H);[M+H]⁺ m/z 431.

Example 5 5-Amino-3-benzyl-3H-thiazolo[4,5-d]pyrimidin-2-one (18)

-   -   a. POCl₃, DMF, 90° C. b. Guanidine HCl, NaOMe, MeOH, 19% over 2        steps

To POCl₃ (8.09 mL, 86.8 mmol) at 0° C. was added consecutively solid3-benzyl-thiazolidine-2,4-dione [prepared according to the method of Lo,et. al. J. Org. Chem. 1957, 999-1001] and DMF. The reaction mixture wasstirred 5 min, then transferred to a 90° C. oil bath where it wasstirred 3 h. The dark mixture was poured into ice (100 g) and water (100mL), and then extracted with EtOAc (3×100 mL). The combined organicphases were dried over MgSO₄ then filtered through celite, concentrated,taken up in DCM, filtered through a short pad of SiO₂ and finallyconcentrated to a dark oil that was used without further purification.

Guanidine hydrochloride (8.87 g, 92.8 mmol) was added as solid to 25%NaOMe-MeOH (18 mL, 79.6 mmol) in MeOH (48 mL) at −5° C. The resultingmixture was stirred 5 min and then treated with crude chloroaldehyde[from above] as a solution in MeOH (20 mL). The reaction mixture wasplaced into a 90° C. oil bath, concentrated by distillation of the MeOHover 2 h, and then heated an additional 30 min. The residue was taken upin EtOAc (200 mL) and partitioned with 1 N HCl (100 mL). The aqueousphase was treated with solid NaHCO₃ and partitioned with EtOAc (3×100mL). The combined organic phases from the alkaline extraction were driedover Na₂SO₄ and then concentrated to a residue that was submitted tochromatography (SiO₂, 50-70% EA-DCM), affording 1.4 g (19%) of the titlecompound 18 as a white solid: ¹H (400 MHz, DMSO-d₆) δ 8.32 (s, 1H),7.24-7.34 (m, 5H), 6.83 (s, 2H), 5.01 (s, 2H); [M+H]⁺ m/z 259.

Example 65-Amino-3-(3′,3′-C,O-dimethyl-β-D-ribofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one(23)

Step 1) Preparation of1,2-O-Isopropylidene-3-methyl-3-O-methyl-5-O-trityl-α-D-ribofuranose(20)

-   -   a. KH (xs), dioxane, DMF (3:1); MeI, rt, 72 h, 87%

To a mixture of tertiary alcohol 19 (716 mg, 1.60 mmol) [preparedaccording to the method of Just et. al. Tetrahedron Lett. 2000, 41,9223-9227] in anhydrous dioxane (6 mL) was added an excess of KH (30%dispersion in mineral oil) at rt. The resulting mixture was stirred 1 hand was then treated with MeI (2 mL, 32 mmol), producing a copiousprecipitate. DMF (2 mL) was added to keep the reaction mixture fluidenough for stirring over 72 h. The mixture was diluted with EtOAc (100mL) and partitioned with saturate aqueous NaHCO₃ (50 mL). The organicphase was dried over Na₂SO₄, concentrated and submitted to flashchromatography (SiO₂, 10-60% EtOAc-hexanes), affording 640 mg of methylether 20 as a white solid: ¹H (400 MHz, DMSO-d₆) δ 7.22-7.35 (m, 15H),5.72 (d, J=2.9, 1H), 4.30 (d, J=2.9, 1H), 4.07 (dd, J=7.7, 2.6, 1H),3.14 (s, 3H), 2.92-3.05 (m, 2H), 1.51 (s, 3H), 1.28 (s, 3H), 0.82 (s,3H).

Step 2) Preparation of1,2,5-tetra-O-Acetyl-3,3-C₁₀-dimethyl-β-D-ribofuranose (21)

To a solution of HOAc (50.0 mL) and Ac₂O (3.83 mL, 40.6 mmol) was addedfuranose 20 (3.62 g, 8.11 mmol). To this colorless solution was added 1M H₂SO₄ in HOAc (0.41 mL, 0.41 mmol), resulting in an intensely yellowcolored solution. The solution was stirred at rt for 16 hr, and thenconcentrated via rotary evaporation. The excess HOAc was azeotroped viaseveral volumes of toluene. The residue was dissolved in DCM (100 mL)and washed with saturated NaHCO₃ (30 mL). The organic layer was driedover Na₂SO₄, decanted, and concentrated to an oily heterogenous mixture.This mixture was subjected to flash chromatography (SiO₂, 5-35%EtOAc-hexanes) affording 0.78 g (33%) of triacetate 21 as a pale yellowoil: ¹H (400 MHz, DMSO-d₆) δ 5.91 (d, J=2.0, 1H), 5.07 (d, J=2.4, 1H),4.29 (dd, J=3.2, 12.0, 1H), 4.15 (dd, J=3.2, 7.2, 1H), 3.96 (dd, J=6.8,12.0, 1H), 3.17 (s, 3H), 2.10 (s, 3H), 2.04 (s, 6H), 1.33 (s, 3H).

Step 3) Preparation of5-Amino-3-(2′,5′-di-O-acetyl-3′,3′-C,O-dimethyl-β-D-ribofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one(22)

To a mixture of heterocycle 1 in anhydrous MeCN (5.0 mL) was addeddropwise BSA (0.52 mL, 2.11 mmol) at rt. The resulting mixture wasimmersed in a 40° C. oil bath, and stirred for 90 min whereupon itbecame homogenous. Furanose 21 (0.20 g, 0.73 mmol) was added followed byTMSOTf (158.4 μL, 0.88 mmol). The reaction mixture was then immersed inan 80° C. oil bath and heated for 2 hr. The reaction was cooled to rt,and partitioned between 1 M pH 7 phosphate buffer (15 mL) and EtOAc (30mL). The resulting emulsion was filtered through a pad of celiteyielding two distinct layers. The organic layer was separated and driedover Na₂SO₄, filtered, and concentrated in vacuo to a dark orange brownresidue. This residue was submitted to flash chromatography (SiO₂, 5-50%EtOAc-hexanes) purification affording 0.30 g (59%) of nucleoside 22 as afinely divided pale yellow solid: ¹H (400 MHz, DMSO-d₆) δ 8.34 (s, 1H),6.90 (br s, 2H), 6.11 (dd, J=8.0, 27.6, 2H), 4.3-4.17 (m, 3H), 2.75 (s,3H), 2.02 (s, 3H), 2.01 (s, 3H), 1.35 (s, 3H); [M+H]⁺ m/z 413.

Step 4) Preparation of5-Amino-3-(3′,3′-C,O-dimethyl-β-D-ribofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one(23)

Nucleoside 22 (230 mg, 0.56 mmol) was dissolved in 5.6 mL MeOH. SolidK₂CO₃ (15.4 mg, 0.11 mmol) was added and the reaction stirred at rt for16 h. HOAc (16.0 μl, 0.28 mmol) was added and the reaction stirred for30 min, and then concentrated mixture to dryness in vacuo. The residualyellow solids were triturated with Et₂O (3×10 mL) carefully decantingfiltrates via pipette. The solid material was then washed with H₂O (3×5mL), rinsed with Et₂O (2×5 mL) and dried on house vacuum 24 h to obtain76.4 mg (42%) of a finely divided white solid: ¹H (400 MHz, DMSO-d₆) δ8.35 (s, 1H), 6.83 (br s, 2H), 5.93 (d, J=8.58, 1H), 5.24 (d, J=7.02,1H), 4.95 (t, J=7.4, 1H), 4.57 (t, J=5.46, 1H), 3.95 (t, J=5.07, 1H),3.46-3.58 (m, 2H), 3.26 (s, 3H), 1.29 (s, 3H); [M+H]⁺ m/z 329.

Example 75-Amino-3-(5′-O-Acetyl-2′-O-[2″-O-acetylpropyl]-3-methyl-β-D-ribofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one(27)

Step 1) Preparation of1,2-O-Isopropylidine-3-methyl-5-O-trityl-α-D-ribofuranose (25)

To a solution of 24 (2.40 g, 5.60 mmol) [prepared according to Bera, et.al. Helvetica Chimica Acta 2000, 83(7), 1398-1407] dissolved in EtOAc(50 mL), under a blanket of N₂, was added 5% Pd/C (240 mg). The flaskwas charged with H₂ at 1 atm, and stirred at rt for 72 h. The reactionmixture was filtered through celite, and concentrated in vacuo to aclear colorless oil. Flash chromatography purification (SiO₂, 0-40%EtOAc-hexanes) afforded 1.88 g of 25 (78%) as a 6:1 (α/β) mixture ofisomers: ¹H (400 MHz, DMSO-d₆) δ 7.22-7.38 (m, 15H), 7.63 (d, J=3.2,1H), 4.53 (t, J=4.0, 1H), 3.67 (dq, J=2.8, 1.6, 1H), 3.18 (dd, J=3.2,10.4, 1H), 2.99 (dd, J=5.2, 10.8, 1H), 1.89-1.98 (m, 1H), 1.38 (s, 3H),1.24 (s, 3H), 0.81 (d, J=6.8, 3H).

Step 2) Preparation of1,5-di-O-Acetyl-2-O-(2′-O-acetylpropyl)-3-methyl-β-D-ribofuranose (26)

In a manner similar to Step 3 of Example 6, 25 was converted to 26 in a19% yield. The crude residue was submitted to flash chromatography(SiO₂, 2-30% EtOAc-hexanes), yielding a 5:1 (β/α) mixture of anomers(tainted with triphenylmethane) by ¹H NMR (DMSO-d₆) that was usedwithout further purification in the next step: ¹H (400 MHz, DMSO-d₆) δ6.07 (d, J=2.8, 1H), 5.02 (ddd, J=6.8, 6.8, 2.8, 1H), 4.25 (dd, J=12.0,3.2, 1H), 4.06-4.21 (m, 2H), 2.14-2.23 (m, 1H), 2.03 (s, 3H), 2.00 (s,3H), 1.99 (s, 3H), 1.41 (s, 3H), 1.39 (s, 3H), 0.91 (d, J=6.8, 3H).

Step 3) Preparation of5-Amino-3-(5′-O-Acetyl-2′-O-[2″-O-acetylpropyl]-3-methyl-β-D-ribofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one(27)

To a mixture of the heterocycle 1 in anhydrous MeCN (5.0 mL) at rt wasadded dropwise BSA (0.52 mL, 2.11 mmol). The resulting mixture stirredfor 30 min, and then furanose 26 (0.20 g, 0.73 mmol) was added followedby TMSOTf (158.4 μL, 0.88 mmol). The reaction mixture was immersed in a60° C. oil bath, whereupon it became a homogenous solution. Stirring wascontinued for 2.5 h. The reaction mixture was cooled to rt, thenpartitioned between 1 M pH 7 phosphate buffer and EtOAc. The mixture wasfiltered through a pad of celite and the distinct layers were separated.The organic phase was dried over Na₂SO₄, filtered, concentrated andsubmitted to flash chromatography (SiO₂, 5-50% EtOAc-hexanes), affording30.0 mg of the title compound 27 as a white solid: ¹H (400 MHz, DMSO-d₆)δ 8.36 (s, 1H), 6.90 (s, 2H), 6.09 (d, J=7.2, 1H), 5.16 (t, J=7.2, 1H),4.88 (dt, J=6.4, 3.2 1H), 4.20 (dd, J=3.2, 12.0, 1H), 4.08 (dd, J=6.4,12.0, 1H), 2.28 (dq, J=6.8, 14, 1H), 1.97 (s, 3H), 1.87 (s, 3H), 1.54(s, 3H), 1.41 (s, 3H), 0.86 (d, J=6.8, 3H); [M+H]⁺ m/z 441.

Example 85-Amino-3-(3′-methyl-β-D-ribofuransyl)-3H-thiazolo[4,5-d]pyrimidin-2-one(30)

Step 1) Preparation of5-Amino-3-(3′-O-acetyl-2′,5′-di-O-benzoyl-3′-methyl-β-D-ribofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one(29)

5-Amino-3H-thiazolo[4,5-d]pyrimidin-2-one (123 mg, 0.733 mmol),3′-C-methyl-ribofuranose 28 [prepared according to the method of Wang etal. J. Med. Chem. 2000, 43, 3704-3713] (302 mg, 0.66 mmol), BSA (447 mg,2.2 mmol) and MeCN (8 mL) were mixed vigorously for 30 min until ahomogeneous solution was obtained. The reaction was then charged withTMSOTf (0.186 mL, 1.1 mmol) and placed into a preheated oil bath at 65°C. After 3 h the reaction was cooled to rt and the solvent was removedby rotary evaporation. The resultant solid was dissolved in EtOAc (200mL) and extracted by saturated aqueous NaHCO₃ (2×100 mL). The organicphase was dried with Na₂SO₄ and concentrated. The crude product wassubmitted to flash chromatography (SiO₂, 0 to 40% EtOAc-CHCl₃), yielding365 mg (88%) of a tan solid: ¹H NMR (400 MHz, d₆-DMSO) δ 8.38 (s, 1H),7.92 (d, J=6.4, 4H), 7.69 (m, 2H), 7.53 (m, 4H), 6.93 (br s, 2H), 6.90(s, 1H), 6.43 (d, J=7.2, 1H), 6.10 (t, J=9.2, 1H), 4.80 (br s, 1H), 4.59(br, s, 1H), 2.09 (s, 3H), 1.97 (s, 3H); [M+H]⁺ m/z 565.

Step 2) Preparation of5-Amino-3-(3′-methyl-β-D-ribofuransyl)-3H-thiazolo[4,5-d]pyrimidin-2-one(30)

5-Amino-3-(2′,5′-di-O-benzoyl-3′-O-acetyl-3′-C-methyl-β-D-ribofuransyl)-3H-thiazolo[4,5-d]pyrimidin-2-one(365 mg, 0.647 mmol) was dissolved in MeOH (10 mL). K₂CO₃ (17.9 mg,0.129 mmol) was added, and the reaction was stirred for 16 h at rt.Acetic acid (15.5 mg, 0.258 mmol) was added, and the reaction wasconcentrated via rotary evaporation. The crude product was thensubmitted to HPLC purification (MeCN—H₂O), yielding 135 mg (66%) of asolid material: ¹H NMR (400 MHz, d₆-DMSO) δ 8.35 (s, 1H), 6.82 (br s,2H), 5.91 (d, J=8.0, 1H), 5.38 (d, J=6.4, 1H), 4.81 (t, J=6.0, 1H), 4.68(s, 1H), 4.51 (t, J=5.6, 1H), 3.78 (t, J=5.6, 1H), 3.48-3.53 (m, 2H),1.21 (s, 3H); [M+H]⁺ m/z 315. Analysis calc'd for C₁₁H₁₄N₄O₅S.0.5H₂O: C,40.86; H, 4.68; N, 17.33; S, 9.92. Found: C, 40.78; H, 4.90; N, 16.94;S, 9.87.

Example 9 Preparation of5-amino-2,3-dihydro-2-thioxo-3-β-D-ribofuranosyl-thiazolo[4,5-d]pyrimidin-7(6H)-one(33)

Step 1) Preparation of5-amino-2,3-dihydro-2-thioxo-3-(2′,3′,5′-tri-O-acetyl-β-D-ribofuranosyl)thiazolo[4,5-d]pyrimidin-7-(6H)-one(32)

Heterocycle 31 [prepared according to the method of Robins et. al. J.Med. Chem. 1990, 33, 407-415] (150 mg, 0.75 mmol), TAR (214 mg, 0.675mmol), MeCN (10 mL) and BSA (0.55 mL, 2.25 mmol) were combined andheated for 1 h at 60′C. The reaction was then charged with TMSOTf (250mg, 1.13 mmol) and stirred for 16 h at 60′C. The mixture wasconcentrated via rotary evaporation, and the crude solid was dissolvedin EtOAc (20 mL). This organic phase was then extracted with saturatedaqueous NaHCO₃ (2×10 mL), and concentrated to dryness by rotaryevaporation. Trituration of the residue with Et₂O (10 mL) yielded 200 mg(80%) of a solid material that was further purified via HPLC (MeCN—H₂O)to generate an analytically pure sample: ¹H NMR (400 MHz, d₆-DMSO) δ11.54 (s, 1H), 7.04 (br s, 2H), 6.59 (m, 1H), 6.10 (m, 1H), 5.70 (t,J=7.2, 1H), 4.42 (dd, J=12.0, 3.2, 1H), 4.27 (m, 1H), 4.18 (m, 1H), 2.08(s, 3H), 2.05 (s, 3H), 1.99 (s, 3H); [M+H]⁺ m/z 459. Analysis calc'd forC₁₆H₁₈N₄O₈S₂: C, 41.92; H, 3.96; N, 12.22; S, 13.99. Found: C, 41.78; H,3.99; N, 12.02; S, 13.72.

Step 2) Preparation of5-amino-2,3-dihydro-2-thioxo-3-β-D-ribofuranosyl-thiazolo[4,5-d]pyrimidin-7(6H)-one(33)

Nucleoside triester 32 (100 mg, 0.21 mmol) and K₂CO₃ (42.7 mg, 0.31mmol) were dissolved in MeOH (5 mL) and stirred for 16 h at ambienttemperature. To this mixture was added HOAc (37 mg, 0.62 mmol) and thesolvent was removed via rotary evaporation. The residue were thensubmitted to HPLC purification (MeCN—H₂O) yielding 47 mg (66%) of asolid: ¹H NMR (400 MHz, d₆-DMSO) δ 11.66 (s, 1H), 6.91 (br s, 2H), 6.47(s, 1H), 5.31 (d, J=5.2, 1H), 4.94 (s, 1H), 4.78 (s, 2H), 4.27 (d,J=8.0, 1H), 3.78 (m, 1H), 3.69 (m, 1H), 3.52 (m, 1H); [M+H]⁺ m/z 333.Analysis calc'd for C₁₀H₁₂N₄O₅S₂.0.5 TFA.0.75H₂O.0.25 MeCN: C, 33.43; H,3.60; N, 14.41. Found: C, 33.11; H, 3.73; N, 14.80.

Example 105-amino-2,3-dihydro-2-thioxo-3-(2′,3′,5′-tri-O-acetyl-β-D-xylofuranosyl)thiazolo[4,5-d]pyrimidin-7-(6H)-one(34)

Preparation of5-amino-2,3-dihydro-2-thioxo-3-(2′,3′,5′)-tri-O-acetyl-β-D-xylofuranosyl)thiazolo[4,5-d]pyrimidin-7-(6H)-one(34)

Heterocycle 31 (265.3 mg, 1.33 mmol), tetraacetylxylofuranose (380 mg,1.19 mmol), BSA (1.26 mL, 5.32 mmol) and MeCN (10 ml) is heated to 60°C. for 30 minutes. Then the reaction was charged with TMSOTf (0.36 mL,2.0 mmol). After 4 h the reaction was worked up by removing the solventby rotary vacuum and taking up the crude solid in ethyl acetate (15 mL).This organic phase was then extracted with saturated sodium bicarbonate(2×10 mL). The organic phase was concentrated and the crude solid wastriturated in 1:1 EtOAc-hexanes. The solid was collected and submittedto HPLC purification (MeCN—H₂O) yielding 40 mg (15%): ¹H NMR (400 MHz,d₆-DMSO) δ 11.45 (s, 1H), 6.90 (br s, 2H), 6.49-6.58 (m, 1H), 6.35-6.49(m, 1H), 5.58 (d, J=5.6, 1H), 4.55 (s, 1H), 4.31 (m, 2H), 2.11 (m, 3H),1.99 (m, 3H), 1.98 (m, 3H); [M+H]⁺ m/z 459. Analysis calc'd forC₁₆H₁₈N₄O₈S₂: C, 41.92; H, 3.96; N, 12.22; S, 13.99. Found: C, 42.14; H,3.99; N, 12.11; S, 14.01.

Example 115-Amino-3-β-D-ribofuranosyl-3H-thiazolo-[4,5-d]pyrimidin-2-thione (39)

Step 1) Preparation of 5-Amino-3H-thiazolo[4,5-d]pyrimidne-2-thione (37)

5-Bromo-pyrimidine-2,4-diamine (2.0 g, 10.58 mmol) [prepared in a mannersimilar to English et. al. J. Am. Chem. Soc. 1946, 68, 453-458] andO-ethylxanthic acid potassium salt (3.39 g, 21.16 mmol) was heated inDMF (25 mL) to 140° C. After 5 h the reaction was cooled to ambienttemperature and 25 mL of water was added. The pH was then adjusted to5.0 using 1 N HCl. A red precipitate formed and was collected byfiltration, yielding 900 mg (30%) of a solid: ¹H NMR (400 MHz, d₆-DMSO)δ 13.85 (s, 1H), 8.33 (br s, 1H), 6.90 (s 2H).

Step 2) Preparation of5-Amino-3-(2′,3′,5′-tri-O-acetyl-β-D-ribofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-thione(38)

5-Amino-3H-thiazolo[4,5-d]pyrimidine-2-thione (250 mg, 1.36 mmol), TAR(389 mg, 1.22 mmol) and BSA (1.0 mL, 4.08 mmol) was heated to 60′C inacetonitrile (10 mL). After 30 minutes the reaction was charged withTMSOTf (0.37 mL, 2.04 mmol) and the reaction was allowed to progress for16 h. The solvent was then removed via rotary evaporation, and the crudeproduct re-dissolved in EtOAc (15 mL). The organic phase was extractedwith concentrated aqueous NaHCO₃ (2×10 mL). The organic phase was thenconcentrated again and submitted to flash chromatography (SiO₂, 5%MeOH-EtOAc) yielding 301 mg (57%) of white solid: ¹H NMR (400 MHz,d₆-DMSO) δ 8.49 (s, 1H), 7.08 (br s, 2H), 6.65 (s 1H), 6.12 (m, 1H),5.79 (t, J=8.0, 1H), 4.43 (dd, J=12.0, 3.6, 1H), 4.28-4.34 (m, 1H), 4.17(dd, J=11.6, 6.8, 1H), 2.09 (s, 3H), 2.05 (s, 3H), 1.97 (s, 3H); [M+H]⁺m/z 443.

Step 3) Preparation of5-Amino-3-β-D-ribofuranosyl-3H-thiazolo-[4,5-d]pyrimidin-2-thione (39)

5-Amino-3-(2′,3′,5′-tri-O-acetyl-β-D-ribofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-thione(202 mg, 0.46 mmol) was dissolved in MeOH (5 mL), and K₂CO₃ (18.9 mg,0.14 mmol) was added. After 1 h acetic acid (21 mg, 0.28 mmol) wasadded, and the reaction was concentrated via rotary evaporation. Thecrude solid was then submitted to HPLC purification (MeCN—H₂O), yielding108 mg (74%) of white solid: ¹H NMR (400 MHz, d₆-DMSO) δ 8.45 (s, 1H),6.96 (br s, 2H), 6.53 (d, J=4.4, 1H), 5.33 (d, J=6.0, 1H), 5.03 (m, 1H),4.86 (d, J=6.4, 1H), 4.67 (t, J=6.0, 1H), 4.33 (m, 1H), 3.79 (m, 1H),3.70 (m, 1H), 3.53 (m, 1H). Analysis calc'd for C₁₀H₁₂N₄O₄S₂.0.35H₂O: C,37.22; H, 3.97; N, 17.36; S, 19.87. Found: C, 37.64; H, 3.87; N, 17.02;S, 19.39.

Example 125-Amino-3-β-D-xylofuranosyl-3H-thiazolo-[4,5-d]pyrimidin-2-thione (41)

Step 1) Preparation of5-Amino-3-(2′,3′,5′-tri-O-acetyl-β-D-xylofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-thione(40)

5-Amino-3H-thiazolo[4,5-d]pyrimidine-2-thione (237 mg, 1.28 mmol),tetra-acetylxylose (370 mg, 1.16 mmol) and BSA (1.25 mL, 5.12 mmol) wereheated in MeCN (10 mL) to 60° C. for 30 min. To this was added TMSOTf(347 μL, 1.92 mmol), and the reaction mixture was stirred at 60° C. for16 h, whereupon the solvent was removed by rotary evaporation, and thecrude solid was re-dissolved in EtOAc (15 mL). This organic phase wasthen extracted with concentrated NaHCO₃ (2×10 mL), and then concentratedto a solid residue that was submitted to flash chromatography (0-100%EtOAc-CHCl₃) yielding 67 mg (13%) of tan solid: ¹H NMR (400 MHz,d₆-DMSO) δ 8.51 (s, 1H), 6.90 (br s, 2H), 6.67 (d, J=4.0, 1H), 6.49 (t,J=2.0, 1H), 5.62 (m, 1H), 5.63 (m, 1H), 4.37 (m, 1H), 4.21 (br m, 1H),2.18 (s, 3H), 1.97 (s, 3H), 1.94 (s, 3H); [M+H]⁺ m/z 443.

Step 2) Preparation of5-Amino-3-β-D-xylofuranosyl-3H-thiazolo-[4,5-d]pyrimidin-2-thione (41)

5-Amino-2,3-dihydro-2-thioxo-3-(2,3,5-tri-O-acetyl-β-D-xylofuranosyl)thiazolo[4,5-d]pyrimidin-7-thione(65 mg, 0.14 mmol) is dissolved in MeOH (5 mL). To this was added K₂CO₃(19 mg, 0.137 mmol), and the resultant mixture was stirred for 3 hwhereupon it was quenched with HOAc (140 μL, 2.4 mmol), and the solventwas removed by rotary evaporation. The crude product is submitted toHPLC purification (MeCN—H₂O) yielding 30 mg (62%) of white solid: ¹H NMR(400 MHz, d₆-DMSO) δ 8.50 (s, 1H), 6.90 (br s, 2H), 6.45 (d, J=5.2, 1H),5.67 (d, J=8.0, 1H), 5.49 (d, J=8.4, 1H), 5.03 (m, 1H), 4.49 (t, J=5.2,1H), 4.02 (m, 2H), 3.72 (m, 2H). Analysis calc'd forC₁₀H₁₂N₄O₄S₂.0.4H₂O: C, 37.12; H, 3.99; N, 17.32; S, 19.82. Found: C,37.53; H, 3.80; N, 17.04; S, 19.42.

Example 135-Amino-7-ethoxy-3-(2′,5′-di-O-acetyl-3′-deoxy-β-D-ribofuranosyl)-3H,6H-thiazolo[4,5-d]pyrimidine-2-one(43)

Step 1) Preparation of5-Amino-7-hydroxy-3-(2′,5′-di-O-acetyl-3′-deoxy-β-D-ribofuranosyl)-3H,6H-thiazolo[4,5-d]pyrimidine-2-one(42)

To a mixture of heterocycle 6 (4.60 g, 25.00 mmol) in anhydrous MeCN(83.0 mL) was added dropwise BSA (15.28 mL, 62.49 mmol). The reactionwas then immersed in a 40° C. oil bath and stirred for 90 min, and1,2,5-tri-O-acetyl-β-D-ribofuranose (5.42 g, 20.80 mmol) was addedfollowed by TMSOTf (5.65 mL, 31.24 mmol). The resulting thick mixturewas immersed in an 80° C. oil bath whereupon the mixture clarified to ahomogenous solution after 15 min. The reaction was stirred for 2 h at80° C., cooled to rt, and then partitioned between 1 M pH 7 phosphatebuffer (50 mL) and EtOAc (100 mL). The resulting emulsion was filteredthrough a pad of celite yielding two distinct layers that wereseparated. The organic layer was dried over Na₂SO₄, filtered, andconcentrated in vacuo to a residue. This residue was submitted to flashchromatography (SiO₂, 0-6% MeOH-DCM) affording 3.41 g (43%) ofnucleoside 42 as a finely divided pale yellow solid: ¹H (400 MHz,DMSO-d₆) δ 11.22 (s, 1H), 6.95 (br s, 2H), 5.79 (d, J=2.0, 1H), 5.59 (d,J=7.2, 1H), 4.20-4.34 (m, 1H), 4.22 (dd, J=3.2, 12.0, 1H), 3.99 (dd,J=6.4, 11.6, 1H), 2.57-2.67 (m, 1H), 2.05 (s, 3H), 1.99 (s, 3H),1.97-2.03 (m, 1H); [M+H]⁺ m/z 385. Analysis calc'd for C₁₄H₁₆N₄O₇S: C,43.75; H, 4.20; N, 14.58; S, 8.34. Found: C, 43.64; H, 4.31; N, 14.37;S, 8.19.

Step 1) Preparation of5-Amino-7-ethoxy-3-(2′,5′-di-O-acetyl-3′-deoxy-β-D-ribofuranosyl)-3H,6H-thiazolo[4,5-d]pyrimidine-2-one(43)

To a solution of 42 (99 mg, 0.26 mmol) dissolved in anhydrous THF (5.5mL) was added S-TPP PPh₃ resin (0.36 g, 0.77 mmol, 2.15 mmol/g). Themixture was chilled to 0° C., and EtOH (30.1 μL, 0.52 mmol) was addedfollowed by DEAD (176.0 μL, 0.39 mmol). The reaction mixture was removedfrom the ice bath and warmed to rt whereupon it stirred for 16 h. Themixture was concentrated in vacuo to a residue that was subjected toseveral passes of flash chromatography purification (SiO₂, elution with2% MeOH/0-40% EtOAc in hexanes) affording 0.22 mg of 43 (20%): ¹H (400MHz, DMSO-d₆) δ 6.95 (br s, 2H), 5.87 (d, J=2.4, 1H), 5.64 (d, J=7.2,1H), 4.39 (q, J=6.8, 2H), 4.32-4.4 (m, 1H), 4.25 (dd, J=3.2, 11.6, 1H),3.96-4.03 (m, 1H), 2.63-2.71 (m, 1H), 2.05 (s, 3H), 2.03-2.08 (m, 1H),1.99 (s, 3H), 1.30 (t, J=6.8, 3H); [M+H]⁺ m/z 413.

Example 145-Amino-7-ethoxy-3-(β-D-ribofuranosyl)-2,3-dihydro-2-thioxo-thiazolo[4,5-d]pyrimidin-7(6H)-one(45)

Step 1) Preparation of5-Amino-7-ethoxy-3-(2′,3′,5′-tri-O-acetyl-β-D-ribofuranosyl)-2,3-dihydro-2-thioxo-thiazolo[4,5-d]pyrimidin-7(6H)-one(44)

5-Amino-2,3-dihydro-2-thioxo-3-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl)thiazolo[4,5-d]pyrimidin-7(6H)-one(250 mg, 0.54 mmol) and S-TPP Ph₃P resin (753 mg, 1.62 mmol) weresuspended in THF (15 mL) and cooled to 0° C. Ethyl alcohol (50 μL, 1.08mmol) and DEAD (148 μL, 0.82 mmol) were added sequentially. After 1 hthe reaction mixture was warmed to ambient temperature and stirred for16 h. The reaction mixture was then filtered, concentrated and submittedto flash chromatography (SiO₂, 15% EtOAc-CHCl₃), affording 200 mg (65%)of a white foam: ¹H NMR (400 MHz, d₆-DMSO) δ 6.91 (s, 1H), 6.47 (br s,2H), 6.29 (m, 1H), 6.18 (s, 1H), 4.62-4.31 (m, 5H), 1.42 (t, J=4.2. 3H),1.38 (s, 3H), 1.35 (s, 3H), 1.32 (s, 3H); [M+H]⁺ m/z 487.

Step 2) Preparation of5-Amino-7-ethoxy-3-β-D-ribofuranosyl-2,3-dihydro-2-thioxo-thiazolo[4,5-d]pyrimidin-7(6H)-one(45)

5-Amino-2,3-dihydro-2-thioxo-3-(2,3,5-tri-O-acetyl-(3-D-ribofuranosyl)thiazolo-[4,5-d]pyrimidin-7(6H)-ethylether (180 mg, 0.37 mmol) and K₂CO₃ (12.8 mg, 0.01 mmol) were suspendedin MeOH (5 mL). After 1 h acetic acid was added and the solvent removedrotary evaporation. The crude product was then submitted to HPLCpurification (MeCN—H₂O) yielding 105 mg (83%) of a solid: ¹H NMR (400MHz, d₆-DMSO) δ 6.98 (s, 2H), 6.50 (d, J=4.8, 1H), 5.32 (d, J=5.2, 1H),5.01 (s, 1H), 4.85 (d, J=5.6, 1H), 4.68 (t, J=5.6, 1H), 4.43 (dd,J=13.6, 6.8, 2H), 4.30 (m, 1H), 3.80 (m, 1H), 3.71 (m, 1H), 3.66 (m,1H), 1.31 (t, J=6.8, 3H); [M+H]⁺ m/z 361.

Preparation of 5-Amino-3H-oxazolo[4,5-d]pyrimidin-2-one (50)

2,4-Diamino-pyrimidin-5-ol (500 mg, 3.97 mmol) [prepared according tothe method of Hull. J. Chem. Soc. 1956, 2033-2035] was suspended in DMF(10 mL). To this was added consecutively NaH (86.7 mg, 3.77 mmol) andCDI (707 mg, 4.36 mmol), and the reaction mixture was heated withvigorous stirring at 60° C. for 3 h. The mixture was cooled to ambienttemperature, and then quenched with water (25 mL). The solvent and waterwere removed via rotary evaporation, and the residue was then trituratedin water (5 mL). The solid was then collected by filtration and dried,affording 230 mg (38%): ¹H NMR (400 MHz, d₆-DMSO) δ 11.90 (br s, 1H),7.72 (br s, 1H), 6.72 (s, 2H); Analysis calc'd for C₅H₄N₄O₂.0.2H₂O: C,38.56; H, 2.85; N, 35.98. Found: C, 39.01; H, 2.71; N, 35.58; [M+H]⁺ m/z153.

The diaminohydroxypyrimidine 46 was reacted with NaH and CDI in DMF toafford heterocycle 50, or with NaH and TCDI in DMF to provideheterocycle 47. Both aminopyrimidines 47 and 50 can be independentlysubmitted to BSA-TMSOTf mediated coupling reactions with anappropriately selected β-D-ribofuranose (wherein R₁, R₂ and R₃ may beindependently acetyl or benzoyl) to give nucleosides 48 and 51respectively. Alkaline methanolysis of 48 and 51 should afforddeprotected nucleosides 49 and 52 respectively.

Example 15 Preparation of5-Amino-3-pyridin-3-ylmethyl-3H-thiazolo[4,5-d]pyrimidin-2-one (54)

Step 1: Preparation of5-Amino-3-pyridin-3-ylmethyl-3H-thiazolo[4,5-d]pyrimidin-2-one (54)

5-Amino-3H-thiazolo[4,5-d]pyrimidin-2-one (107 mg, 0.64 mmol) wasdissolved in DMF (4 mL) at ambient temperature. Sodium hydride (30 mg,1.32 mmol) was added and the mixture was heated to 30° C. Stirring wascontinued for 0.5 h before 3-bromomethyl-pyridine hydrobromide (179 mg,0.71 mmol) was added. The mixture was then heated to 75° C. and allowedto stir for 4 h. Upon completion, the reaction was allowed to cool toroom temperature, then concentrated. Water (12 mL) was added. Theresulting mixture was diluted with H₂O (12 mL), then extracted withethyl acetate (3×5 mL). The combined organic layers were washed withbrine, dried over Na₂SO₄, filtered, and concentrated. The crude materialwas purified by column chromatography (SiO₂, 20-50% EtOAc-CH₂Cl₂) togenerate 90 mg (54%) of 54 as a white solid: ¹H NMR (400 MHz, DMSO-d₆) δ8.59 (s, 1H), 8.48 (d, J=3.6, 1H), 8.32 (s, 1H), 7.71 (d, J=8.4, 1H),7.36 (m, 1H), 6.86 (s, 2H), 5.04 (s, 2H); [M+H]⁺ 260.1.

Example 16 Preparation of5-Amino-3-(6-chloro-pyridin-3-ylmethyl)-3H-thiazolo[4,5-d]pyrimidin-2-one(55)

Step 1: Preparation of5-Amino-3-(6-chloro-pyridin-3-ylmethyl)-3H-thiazolo[4,5-d]pyrimidin-2-one(55)

In a manner similar to Example 15, Step 1, 111 mg of the title compound55 was generated in 54% yield as an orange solid: ¹H NMR (400 MHz,DMSO-d₆) δ 8.42 (s, 1H), 8.30 (s, 1H), 7.77 (d, J=8.8, 1H), 7.47 (d,J=8.0, 1H), 6.85 (s, 2H), 5.11 (s, 2H); [M+H]⁺ 294.1.

Example 17 Preparation of(Z)-5-Amino-3-(4-chloro-2-buten-1-yl)-3H-thiazolo[4,5-d]pyrimidin-2-one(56)

Step 1: Preparation of(Z)-5-Amino-3-(4-chloro-2-buten-1-yl)-3H-thiazolo[4,5-d]pyrimidin-2-one(56)

In a manner similar to Example 15, Step 1, 74 mg of the title compound56 was generated in 46% yield as a yellow solid: ¹H NMR (400 MHz,DMSO-d₆) δ 8.29 (s, 1H), 6.80 (s, 2H), 5.81 (m, 1H), 5.66 (m, 1H), 4.53(d, J=6.0, 2H), 4.27 (d, J=8.0, 2H); [M+H]⁺ 257.2.

Example 18 Preparation of5-Amino-3-hexyl-3H-thiazolo[4,5-d]pyrimidin-2-one (57)

Step 1: Preparation of 5-Amino-3-hexyl-3H-thiazolo[4,5-d]pyrimidin-2-one(57)

In a manner similar to Example 15, Step 1, 51 mg of the title compound57 was generated in 15% yield as an off-white solid: ¹H NMR (400 MHz,DMSO-d₆) δ 8.28 (s, 1H), 6.78 (s, 1H), 3.82 (t, J=7.2, 2H), 1.64 (m,2H), 1.27 (m, 6H), 0.85 (t, J=6.8, 3H); [M+H]⁺] 253.1.

Example 19 Preparation of(±)-5-Amino-3-cyclopentyl-3H-thiazolo[4,5-d]pyrimidin-2-one (58)

Step 1: Preparation of(±)-5-Amino-3-cyclopentyl-3H-thiazolo[4,5-d]pyrimidin-2-one (58)

In a manner similar to Example 15, Step 1, 21 mg the title compound 58was generated in 5% yield as a light yellow solid: ¹H NMR (400 MHz,CDCl₃) δ 8.09 (s, 1H), 5.23 (s, 2H), 2.24 (m, 1H), 2.00 (m, 4H), 1.66(m, 4H); [M+H]⁺ 237.0.

Example 20 Preparation of5-Amino-3-(4-nitro-phenyl)-3H-thiazolo[4,5-d]pyrimidin-2-one (59)

Step 1: Preparation of5-Amino-3-(4-nitro-phenyl)-3H-thiazolo[4,5-d]pyrimidin-2-one (59)

In a manner similar to Example 15, Step 1, 45 mg of the title compound59 was generated in 10% yield as an orange solid: ¹H NMR (400 MHz,DMSO-d₆) δ 8.59 (s, 1H), 8.18 (d, J=9.2, 2H), 7.99 (d, J=9.2, 2H), 5.74(s, 2H); [M+H]⁺ 290.2.

Example 215-Amino-3-(2,3,5,6-tetrafluoro-pyridin-4-yl)-3H-thiazolo[4,5-d]pyrimidin-2-one(60)

Step 1: Preparation of5-Amino-3-(2,3,5,6-tetrafluoro-pyridin-4-yl)-3H-thiazolo[4,5-d]pyrimidin-2-one(60)

In a manner to Example 15, Step 1, 35 mg of the title compound 60 wasgenerated in 5% yield as an orange solid: ¹H NMR (400 MHz, DMSO-d₆) δ8.16 (s, 1H), 4.01 (s, 2H); [M+H]⁺ 318.4.

Example 22 Preparation of(E)-5-Amino-3-(4-chloro-2-buten-1-yl)-3H-thiazolo[4,5-d]pyrimidin-2-one(62) Step 1: Preparation of(E)-5-Amino-3-(4-chloro-2-buten-1-yl)-3H-thiazolo[4,5-d]pyrimidin-2-one(61)

The title compound 61 can be synthesized by treating5-Amino-3H-thiazolo[4,5-d]pyrimidin-2-one (1) in DMF with sodium hydrideand (E)-1,4-dichloro-2-butene under various conditions.

Step 2: Preparation of(E)-5-Amino-3-(4-hydroxy-2-buten-1-yl)-3H-thiazolo[4,5-d]pyrimidin-2-one(62)

The title compound 62 could be synthesized by treating(E)-5-Amino-3-(4-chloro-2-buten-1-yl)-3H-thiazolo[4,5-d]pyrimidin-2-one(61) with 0.1 M HCl under various conditions.

Example 23 Preparation of(3′S)-5-Amino-3-(3′-deoxy-3′-hydroxymethyl-β-D-ribofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one(65)

Step 1: Preparation of(3′S)-5-Amino-3-(3′-acetoxymethyl-2′,5′-di-O-acetyl-3′-deoxy-β-D-ribofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one(64)

(3S)-3-O-Acetoxymethyl-1,2,5-tri-O-acetyl-3-deoxy-α,β-D-ribofuranose(63) [prepared according to the method of Cooperwood et al. Nucleosides,Nucleotides, and Nucleic Acids 2000, 19, 219-236 in which the enantiomerof the same compound was made] (176 mg, 0.53 mmol) was dissolved inacetonitrile (7 mL) at ambient temperature.5-Amino-3H-thiazolo[4,5-d]pyrimidin-2-one (1) (89 mg, 0.53 mmol) wasadded, the mixture was then stirred for 0.5 h before it was heated to40° C. After 5 min at 40° C., BSA (0.39 mL, 1.59 mmol) was added and themixture was stirred for another 0.5 h. The mixture was then heated to80° C. TMSOTf (0.14 mL, 0.80 mmol) was added and the reaction wasstirred for 3-4 hours at 80° C. Upon completion, the reaction wasallowed to cool to room temperature and then quenched by a pH 7.0 buffer(1.0 M K₂HPO₄ and 1.0 M NaH₂PO₄, 2 ml). The mixture was extracted withCH₂Cl₂ (3×10 mL). The combined organic layers were washed with brine,dried with Na₂SO₄, and concentrated in vacuo. The crude product waspurified by column chromatography (SiO₂, 0-10% MeOH—CH₂Cl₂ to afford 77mg (33%) of 64 as a powdery light yellow solid: ¹H NMR (400 MHz, CDCl₃)δ 8.14 (s, 1H), 6.04 (d, J=1.6, 1H), 5.90 (dd, J=6.8, 1.6, 1H), 5.24 (s,2H), 4.52 (dd, J=12.0, 2.8, 1H), 4.36 (m, 2H), 4.17 (m, 2H), 3.54 (m,1H), 2.18 (s, 9H); [M+H]⁺ 441.2; Elemental analysis forC₁₇H₂₀N₄O₈S.0.6H₂O: calc'd: C, 45.25; H, 4.74; N, 12.42; S, 7.11. found:C, 45.24; H, 4.66; N, 12.02; S, 7.24.

Step 2: Preparation of(3′S)-5-Amino-3-(3′-deoxy-3′-hydroxymethyl-β-D-ribofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one(65)

(3′S)-5-Amino-3-(3′-acetoxymethyl-2′,5′-di-O-acetyl-3′-deoxy-β-D-ribofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one64 (114 mg, 0.28 mmol) was dissolved in methanol (2 mL) at ambienttemperature. Potassium carbonate (2 mg, cat.) was added and the mixturewas stirred at room temperature overnight. Upon completion, acetic acidwas added (2 μL) and the mixture was stirred another 30 minutes at roomtemperature. The mixture was concentrated, purified by HPLC, thentriturated by EtOAc to afford 79 mg (90%) of 65 as a white solid: ¹H NMR(400 MHz, D₂O) δ 8.28 (s, 1H), 6.10 (m, 1H), 5.18 (m, 1H), 4.20 (m, 1H),3.95 (m, 2H), 3.78 (m, 2H), 3.00 (m, 1H); [M+H]⁺ 315.2; Elementalanalysis for C₁₁H₁₄N₄O₅S 0.3H₂O.0.15iPrOH: calc'd: C, 41.83; H, 4.84; N,17.04; S, 9.75. found: C, 41.92; H, 4.61; N, 16.89; S, 9.78.

Example 24 Preparation of5-Amino-3-(5′-deoxy-5′-hydroxymethyl-β-D-ribofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one(68)

Step 1: Preparation of5-Amino-3-(5′-O-acetoxymethyl-2′,3′-di-O-acetyl-5′-deoxy-β-D-ribofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one(67)

In a manner similar to Example 23, Step 1, 113 mg of the title compound67 was generated from5-O-acetoxymethyl-1,2,3-tri-O-acetyl-5-deoxy-α,β-D-ribofuranose (66)[prepared according to the method of Pakulski et al. Polish J. Chem.1995, 69, 912-917] in 53% yield as a sticky yellow solid: ¹H NMR (400MHz, CDCl₃) δ 8.15 (s, 1H), 6.34 (m, 1H), 6.25 (d, J=6.0, 1H), 6.11 (d,J=4.0, 1H), 6.04 (m, 1H), 5.76 (t, J=6.0, 1H), 5.42 (s, 1H), 4.93 (m,1H), 4.35 (m, 1H), 4.21 (q, J=5.6, 1H), 2.20 (s, 9H); [M+H]⁺ 441.2.

Step 2: Preparation of5-Amino-3-(5′-deoxy-5′-hydroxymethyl-β-D-ribofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one(68)

In a manner similar to Example 23, Step 2, 43 mg of the title compound68 was generated in 71% yield as a white solid: ¹H NMR (400 MHz,d₆-DMSO) δ 8.33 (s, 1H), 6.84 (s, 2H), 5.86 (d, J=4.4, 1H), 5.26 (d,J=5.2, 1H), 4.93 (m, 1H), 4.74 (q, J=10.0, 4.4, 2H), 4.40 (m, 1H), 3.82(m, 2H), 1.76 (m, 3H); [M+H]⁺ 315.2; Elemental Analysis forC₁₁H₁₄N₄O₅S.0.4H₂O.0.2iPrOH: calc'd: C, 41.77; H, 4.96; N, 16.80; S,9.61. found: C, 41.61; H, 4.85; N, 16.68; S, 9.58.

Example 25 Preparation of5-Amino-3-(3′-deoxy-3′-O-p-toluenesulfonyl-β-D-xylofuranosyl)-3H-thiazolo-[4,5-d]pyrimidine-2-one(73)

Step 1: Preparation of1,2-O-Isopropyliene-3-O-p-toluenesulfonyl-5-O-trityl-β-D-xylofuranose(70)

1,2-O-Isopropylidene-5-O-trityl-β-D-xylofuranose (69) [preparedaccording to the method of Johnston et al. Tetrahedron Lett. 1995, 36,4341-4344] (4.25 g, 9.83 mmol) was dissolved in pyridine (60 mL) atambient temperature. P-Toluenesulfonyl chloride (2.81 g, 14.74 mmol) wasadded to the solution. After 24 h the reaction had gone to completion,the crude mixture was concentrated. The residue was dissolved in EtOAc(50 mL), washed with saturated aqueous NH₄Cl (25 mL), saturated aqueousNaHCO₃ (25 mL) and brine (25 mL). The organic phase was dried overMgSO₄, filtered, then concentrated. The mixture was then purified byISCO chromatography (SiO₂, 2-15% EtOAc-Hexane), affording 5.20 g (90%)of 70 as a white solid: ¹H (400 MHz, CDCl₃) δ 7.61 (m, 2H), 7.32-7.34(m, 6H), 7.23-7.32 (m, 9H), 5.92 (d, J=4.4, 1H), 4.74 (dd, J=11.2, 3.6,2H), 4.19-4.22 (m, 1H), 3.45 (dd, J=10.4, 6.4, 1H), 3.05 (q, J=5.2, 1H),2.40 (s, 3H), 1.49 (s, 3H), 1.31 (s, 3H).

Step 2: Preparation of1,2,5-Tri-O-acetyl-3-O-p-toluenesulfonyl-α,β-D-xylofuranose (71)

1,2-O-isopropyliene-3-O-p-toluenesulfonyl-5-O-trityl-β-D-xylofuranose(70) (5.20 g, 8.86 mmol) was dissolved in AcOH (60 mL) at ambienttemperature. Acetic anhydride (4.23 mL, 44.71 mmol) was added dropwiseto the solution. The resulting mixture was cooled to 0° C., followed byslow addition of 1M H₂SO₄ (9.75 mL, 9.75 mmol). After 24 h the reactionhad gone to completion, the crude mixture was concentrated, thenazeotroped with toluene (2×20 mL). The residue was dissolved in CH₂Cl₂(50 mL), washed with saturated aqueous NaHCO₃ (20 mL). The Organic phasewas dried over MgSO₄, filtered, then concentrated. The mixture was thenpurified by ISCO chromatography (SiO₂, 2-40% EtOAc-Hexane), affording3.09 g (81%) of 71 as a colorless oil: ¹H (400 MHz, CDCl₃) δ (a mixtureof α and β isomers) 7.80-7.85 (m), 7.37-7.39 (m), 6.36 (d, J=4.4), 6.06(s), 5.20-5.30 (m), 4.56-4.62 (m), 4.26-4.29 (m), 2.50 (s), 2.06-2.08(m).

Step 3: Preparation of5-Amino-3-[2′5′-di-O-acetyl-3′-deoxy-3′-O-p-toluenesulfonyl-β-D-xylofuranosyl]-3H-thiazolo-[4,5-d]pyrimidine-2-one(72)

In a manner similar to Example 23, Step 1, 161 mg of the title compound72 was generated in 54% yield as a fluffy yellow solid: ¹H NMR (400 MHz,CDCl₃) δ 8.12 (s, 1H), 7.85 (d, J=8.8, 2H), 7.39 (d, J=8.8, 2H), 6.18(d, J=2.8, 1H), 5.90 (br s, 2H), 5.77 (d, J=4.4, 1H), 5.01 (dd, J=6.4,5.2, 1H), 4.34 (m, 1H), 4.27 (m, 2H), 2.48 (s, 3H), 2.04 (s, 6H); [M+H]⁺539.3.

Step 4: Preparation of5-Amino-3-[3′-deoxy-3′-O-p-toluenesulfonyl-β-D-xylofuranosyl]-3H-thiazolo-[4,5-d]pyrimidine-2-one(73)

In a manner similar to Example 23, Step 2, 68 mg of the title compound73 was generated in 61% yield as a white solid: ¹H NMR (400 MHz,DMSO-d₆) δ 8.36 (s, 1H), 7.83 (d, J=8.0, 2H), 7.48 (d, J=8.0, 2H), 6.80(s, 2H), 5.92 (d, J=6.0, 1H), 5.71 (d, J=6.4, 1H), 5.20 (m, 1H), 4.89(q, J=5.6, 3.6, 1H), 4.73 (s, 2H), 4.10 (m, 2H), 2.43 (s, 3H); [M+H]⁺455.2; Elemental analysis for (C₁₇H₁₈N₄O₇S₂.0.4H₂O): calc'd: C, 44.22;H, 4.10; N, 12.14; S, 13.89. found: C, 44.45; H, 4.15; N, 12.07; S,13.71.

Example 26 Preparation of5-Amino-3-(3′-deoxy-3′-methylidene-β-D-ribofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one(76)

Step 1: Preparation of5-Amino-3-(2′-O-acetyl-5′-O-benzoyl-3′-deoxy-3′-methylidene-β-D-ribofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one(75)

In a manner similar to Example 23, Step 1, 107 mg of the title compound75 was generated from1,2-di-O-acetyl-5-O-benzoyl-3-deoxy-3-methylidene-α,β-D-ribofuranose(74) [prepared according to the method of Girardet et al. J. Med. Chem.2000, 43, 3704-3713] in 85% yield as a yellow solid: ¹H NMR (400 MHz,CDCl₃) δ 8.13 (s, 1H), 8.05 (dd, J=8.4, 1.2, 2H), 7.57 (tt, J=7.2, 1.2,1H), 7.44 (t, J=7.2, 2H), 6.51 (m, 1H), 6.17 (d, J=4.4, 1H), 5.30 (s,2H), 5.11 (m, 2H), 4.82 (dd, J=11.6, 4.8, 2H), 4.52 (dd, J=11.6, 6.8,1H), 2.14 (s, 3H); [M+H]⁺ 443.2.

Step 2: Preparation of5-Amino-3-(3′-deoxy-3′-methylidene-β-D-ribofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one(76)

In a manner similar to Example 23, Step 2, 35 mg of the title compound76 was generated in 35% yield as a gray-white solid: ¹H NMR (400 MHz,DMSO-d₆) δ 8.37 (s, 1H), 5.83 (d, J=5.6, 1H), 5.74 (d, J=7.6, 1H), 5.51(m, 2H), 5.19 (d, J=11.2, 2H), 4.72 (t, J=6.0, 1H), 4.54 (br s, 2H),3.85 (s, 2H); [M+H]⁺ 297.2; Elemental analysis for(C₁₁H₁₂N₄O₄S.0.2H₂O.0.25iPrOH): calc'd: C, 44.81; H, 4.61; N, 17.79; S,10.18. found: C, 44.84; H, 4.33; N, 17.76; S, 10.22.

Example 27 Preparation of(3′R)-5-Amino-3-(3′-deoxy-3′-fluoro-β-D-xylofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one(79)

Step 1: Preparation of(3′R)-5-Amino-3-(2′-O-acetyl-5′-O-benzoyl-3′-deoxy-3′-fluoro-β-D-xylofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one(78)

In a manner similar to Example 23, Step 1, 148 mg of the title compound78 was generated from1,2-Di-O-acetyl-5-O-benzoyl-3-deoxy-3-(R)-fluoro-α,β-D-xylofuranose (77)[prepared according to the method of Gosselin et al. CarbohydrateResearch 1993, 249, 1-17] in 56% yield as a light yellow solid: ¹H NMR(400 MHz, DMSO-d₆) δ 8.16 (s, 1H), 8.05 (d, J=8.8, 2H), 7.57 (t, J=7.6,1H), 7.44 (t, J=8.0, 2H), 6.29 (ddd, J=21.2, 4.8, 1.2, 1H), 5.98 (d,J=4.8, 1H), 5.32 (ddd, J=52.0, 4.0, 1.2, 1H), 5.20 (s, 1H), 4.83 (dd,J=11.2, 4.8, 1H), 4.61 (m, 1H), 2.00 (s, 3H); [M+H]⁺ 449.3.

Step 2:(3′R)-5-amino-3-(3′-deoxy-3′-fluoro-β-D-xylofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one(79)

In a manner similar to Example 23, Step 2, 43 mg of the title compound79 was generated in 56% yield as yellow solid: ¹H NMR (400 MHz, DMSO-d₆)δ 8.36 (s, 1H), 6.87 (s, 2H), 5.97 (d, J=4.8, 1H), 5.73 (d, J=5.6, 1H),5.22 (dtd, J=24.4, 5.6, 2.0, 1H), 5.02 (ddd, J=52.8, 4.4, 1.6, 1H), 4.07(m, 2H), 3.62 (m, 2H); [M+H]⁺ 303.6.

Example 28 Preparation of(3′S)-5-amino-3-(2′,5′-di-O-acetyl-3′-azido-3′-deoxy-β-D-ribofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one(83)

Step1: Preparation of(3R)-3-Azido-3-deoxy-1,2-O-isopropyliene-5-O-trityl-β-D-ribofuranose(81)

1,2-O-Isopropylidene-5-O-trityl-β-D-xylofuranose (69) [preparedaccording to the method of Johnston et al. Tetrahedron Lett. 1995, 36,4341-4344] (3.28 g, 7.58 mmol) was dissolved in CH₂Cl₂ (75 mL) atambient temperature before it was cooled to −10° C. Pyridine (0.86 mL,10.61 mmol) was added to the solution, followed by slow addition oftrifluoromethanesulfonic anhydride (1.53 mL, 9.10 mmol). After stirringat −10° C. for 1 h the reaction was quenched by slow addition of 5%NaHSO₃ (150 mL) before it was warmed up to room temperature. The layerswere then separated, and the aqueous phase was further extracted withCH₂Cl₂ (2×75 mL). The organic layers were combined, dried with MgSO₄,then filtered and concentrated. The residue was azeotroped with toluene(2×10 mL), then dried on high vacuum to afford triflate 80.

Triflate 80 was dissolved in DMF (100 mL) at ambient temperature.Pyridine (0.92 mL, 11.37 mmol) was added to the solution, followed byaddition of sodium azide (1.97 g, 30.32 mmol). After 5 d the reactionhad gone to completion, the crude mixture was concentrated. The residuewas dissolved in EtOAc (60 mL), washed with saturated aqueous NH₄Cl (40mL). The organic layer was dried over MgSO₄, filtered and concentrated.The mixture was then purified by ISCO chromatography (SiO₂, 2-15%EtOAc-Hexane), affording 1.80 g (52% for 2 steps) of 81 as a whitesolid: ¹H (400 MHz, CDCl₃) δ 7.44-7.47 (m, 6H), 7.26-7.32 (m, 6H),7.24-7.25 (m, 3H), 5.90 (d, J=3.6, 1H), 4.77 (t, J=4.4, 1H), 4.18-4.22(m, 1H), 3.66 (q, J=6.0, 1H), 3.52 (dd, J=10.4, 3.2, 1H), 3.20 (dd,J=10.8, 4.0, 1H), 1.59 (s, 3H), 1.40 (s, 3H).

Step 2: Preparation of(3R)-1,2,5-Tri-O-acetyl-3-azido-3-deoxy-α,β-D-ribofuranose (82)

(3R)-3-Azido-3-deoxy-1,2-O-isopropyliene-5-O-trityl-β-D-ribofuranose(81) (1.20 g, 2.62 mmol) was dissolved in AcOH (30 mL) at ambienttemperature. Acetic anhydride (1.24 mL, 13.10 mmol) was added dropwiseto the solution. The resulting mixture was cooled to 0° C., followed byslow addition of 1M H₂SO₄ (2.88 mL, 2.88 mmol). After 24 h the reactionhad gone to completion, the crude mixture was concentrated, thenazeotroped with toluene (2×10 mL). The residue was dissolved in CH₂Cl₂(30 mL), washed with saturated aqueous NaHCO₃ (20 mL). The Organic phasewas dried over MgSO₄, filtered, and concentrated. The mixture was thenpurified by ISCO chromatography (SiO₂, 2-40% EtOAc-Hexane), affording0.66 g (83%) of 82 as a colorless oil: ¹H (400 MHz, CDCl₃) δ (a mixtureof α and β isomers) 6.43 (d, J=4.4), 6.14 (s), 5.34 (d, J=4.8), 5.21(dd, J=7.6), 4.20-4.37 (m), 4.04-4.10 (m), 2.10-2.20 (m).

Step 3: Preparation of(3′R)-5-amino-3-(2′,5′-di-O-acetyl-3′-azido-3′-deoxy-β-D-ribofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one(83)

In a manner similar to Example 23, Step 1, 288 mg of the title compound83 was generated in 85% yield as a orange solid: ¹H NMR (400 MHz, CDCl₃)δ 8.17 (s, 1H), 6.18 (d, J=2.4, 1H), 5.95 (dd, J=6.4, 2.8, 1H), 5.14 (s,2H), 4.61 (m, 1H), 4.24 (dd, J=12.0, 5.2, 1H), 4.17 (m, 2H), 2.12 (s,6H); [M+H]⁺ 410.4.

Example 29 Preparation of(1′R,2′S,3′R,4′R)—N′-[7-chloro-2-oxo-3-(2′,3′-O-isopropylidene-4′-vinyl-cyclopentan-1′-yl)-2,3-dihydro-thiozolo[4,5-d]pyrimidin-5-yl]-N,N-dimethyl-formamidine(88)

Step 1: Preparation of(1′R,2′S,3′R,4′R)—N′-[7-chloro-2-oxo-3-(2′,3′-O-isopropylidene-4′-vinyl-cyclopentan-1′-yl)-2,3-dihydro-thiozolo[4,5-d]pyrimidin-5-yl]-N,N-dimethyl-formamidine(85)

(1R,2S,3R,4R)-2,3-O-isopropylidene-4-vinyl-cyclopentan-1-ol (84)[prepared according to the method of Yang et al. J. Org. Chem. 2004, 69,3993-3996] (96 mg, 0.52 mmol) was dissolved in CH₂Cl₂ (2 mL) andpyridine (10 mL) at ambient temperature. The solution was cooled to 0°C., followed by slow addition of trifluoromethanesulfonic anhydride (115mL, 0.68 mmol). After 0.5 h, the reaction had gone to completion, thereation was quenched with H₂O (10 mL), then further diluted with CH₂Cl₂(10 mL). After the layers were separated, the aqueous phase was furtherwashed with CH₂Cl₂ (2×10 mL). The Organic fractions were combined, driedover MgSO₄, filtered, then concentrated. The resulting yellowish oil wasused for next step directly.

The above triflate (131 mg, 0.51 mmol) was suspended in CH₃CN (10 mL) atambient temperature. Sodium hydride (15 mg, 0.62 mmol) was added to thesolution, followed by the addition of a solution ofN′-[7-chloro-2-oxo-2,3-dihydro-thiozolo[4,5-d]pyrimidin-5-yl)-N,N-dimethyl-formamidine(Chloroamidine base, 160 mg, 0.62 mmol) in CH₃CN (8 mL). The reactionwas stirred at 50° C. for 12 h before it was quenched by addition of H₂O(5 mL). The resulting mixture was extracted with EtOAc (3×20 mL). Theorganic fractions were combined, dried over MgSO₄, filtered, thenconcentrated. The mixture was then purified by column chromatography(SiO₂, 2-20% EtOAc-Hexane), affording 94.8 mg (43%) of 85 as a whitesolid: ¹H (400 MHz, CDCl₃) δ 8.63 (s, 1H), 5.92 (m, 1H), 5.09-5.29 (m,4H), 4.55-4.61 (m, 1H), 3.23 (s, 3H), 3.21 (s, 3H), 2.65-2.77 (m, 1H),2.42-2.51 (m, 1H), 2.18-2.22 (m, 1H), 1.56 (s, 3H), 1.29 (s, 3H); [M+H]⁺424.1.

Step 2: Preparation of(1′R,2′S,3′R,4′R)-5-Amino-7-chloro-3-(2′,3′-O-isopropylidene-4′-hydroxymethyl-cyclopentan-1′-yl)-3H-thiazolo[4,5-d]pyrimidin-2-one(86)

The title compound 86 can be synthesized by first treating(1′R,2′S,3′R,4′R)—N′-[7-chloro-2-oxo-3-(2′,3′-O-isopropylidene-4′-vinyl-cyclopentan-1′-yl)-2,3-dihydro-thiozolo[4,5-d]pyrimidin-5-yl]-N,N-dimethyl-formamidine(85) in CH₃OH and H₂O with sodium periodate and Osmium tetroxide. Thecrude product can then be treated with sodium borohydride in CH₃OH toafford 86.

Step 3: Preparation of(1′R,2′S,3′R,4′R)-5-Amino-3-(2′,3′-O-isopropylidene-4′-hydroxymethyl-cyclopentan-1′-yl)-3H-thiazolo[4,5-d]pyrimidin-2-one(87)

The title compound 87 can be synthesized by treating(1′R,2′S,3′R,4′R)-5-Amino-7-chloro-3-(2′,3′-O-isopropylidene-4′-hydroxymethyl-cyclopentan-1′-yl)-3H-thiazolo[4,5-d]pyrimidin-2-one(86) in AcOH with Zinc copper couple under various conditions.

Step 4: Preparation of(1′R,2′S,3′R,4′R)-5-Amino-3-(2′,3′-dioxy-4′-hydroxymethyl-cyclopentan-1′-yl)-3H-thiazolo[4,5-d]pyrimidin-2-one(88)

The title compound 88 can be synthesized by treating(1′R,2′S,3′R,4′R)-5-Amino-3-(2′,3′-O-isopropylidene-4′-hydroxymethyl-cyclopentan-1′-yl)-3H-thiazolo[4,5-d]pyrimidin-2-one(87) in CH₃OH with 2M HCl under various conditions.

Example 30 Preparation of5-Amino-3-(3′-(R)-methoxy-β-D-xylofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one(89)

The required sugar, acetic acid1,2,5-tri-O-acetyl-3)-methoxy-D-xylofuranose α and β mixture (91) wasprepared as follows:

Step 1: Preparation of1,2-O-isopropylidene-3-methoxy-5-O-trityl-D-xylofuranose (90)

Trityl alcohol 69 (5 g, 11.57 mmol) was mixed with methyl iodide (2.5ml, 34.7 mmol) in THF (40 ml). Tetrabutylammonium iodide (427 mg) wasadded and the mixture cooled in an ice bath. Under a slow stream ofnitrogen solid sodium hydride-oil mixture (1.33 g, 60% NaH, 34.7 mmol)was added in small portions. The reaction was stirred overnight whilewarming slowly to ambient temperature. The reaction was carefully pouredinto a mixture of saturated ammonium chloride and ice and extractedthree times with ethyl ether. The ether portions were combined, washedwith brine, dried (MgSO₄), filtered and the solvent evaporated to yield90 as a cloudy oil. ¹H NMR (400 MHz, CDCl₃) δ 7.42 7.42 (m, 6H), 7.26(m, 9H), 5.85 (d, J=3.6 Hz, 1H), 4.54 (d, J=3.6 Hz, 1H), 4.38 (m, 1H),3.785 (d, J=3.2 Hz, 1H), 3.42 (m, 1H), 3.35 (m, 4H), 1.53 (s, 3H), 1.336(s, 3H)

Step 2: 1,2,5-tri-O-acetyl-3-methoxy-D-xylofuranose α and β mixture (91)

The trityl compound 90 (6.1 g, 11.57 mmol) was dissolved in a mixture ofacetic acid (20 ml) and acetic anhydride (10 ml) and cooled in a coolwater bath. A mixture of sulfuric acid in acetic anhydride and aceticacid was added (0.5 ml sulfuric acid, 2.5 ml acetic acid, 2.5 ml aceticanhydride, pre-cooled in an ice bath before addition) and the mixturestirred at ambient temperature over night. The reaction was poured onto400 g of ice water and extracted three times with ethyl acetate. Theorganic portions were combined, washed with saturated sodiumbicarbonate, dried (MgSO₄), filtered and evaporated to yield asemisolid. This was purified using flash chromatography on a 120 gsilica gel column eluting with a gradient of ethyl acetate in hexane(10-100%) to give 91 (1.26 g, 4.34 mmol, 38%) as an oily mixture ofanomers. ¹H NMR (400 MHz, CDCl₃) δ 6.39 (d, J=4.4 Hz), 6.17 (s),4.4-4.52 (m), 4.3-4.39 (m), 4.1-4.24 (m), 3.86 (d, J=5.6 Hz), 3.45 (s),3.41 (s), 2.07-2.16 (m).

Step 3: Preparation of5-Amino-3-(3′-methoxy-β-D-xylofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one(89)

In a manner similar to Example 23, step1, using1,2,5-tri-O-acetyl-3-methoxy-D-xylofuranose α and β mixture (91),afforded 43 mg (6%) of 89 as a white solid: ¹H NMR (DMSO-d₆) δ 2.01 (d,J=9.2 Hz, 6H), 3.36 (s, 3H), 4.17-4.24 (m, 2H), 4.31-4.37 (m, 2H), 5.86(d, J=6 Hz, 1H), 6.14 (dd, J=4.4, 1.6 Hz, 1H), 6.85 (br s, 2H), 8.35 (s,1H); MS (ESI) [(M+H)⁺] 399.96, Elemental analysis for(C₁₅H₁₈N₄O₇S.0.5H₂O): calc'd: C, 44.22; H, 4.70; N, 13.75. Found C,44.27; H, 4.54; N, 13.60.

Example 31 Preparation of5-Amino-3-(3′-octyloxy-β-D-xylofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one(92)

The required sugar acetic acid1,2,5-tri-O-acetyl-3(S)-octyloxy-D-xylofuranose α and β mixture (94) wasprepared as follows:

Step 1: Preparation of1,2-O-isopropylidene-3-octyloxy-5-O-trityl-D-xylofuranose (5)

Trityl alcohol 69 (5 g, 11.57 mmol) was mixed with octylbromide (3.99ml, 23.14 mmol) in THF (40 ml). Tetrabutylammonium iodide (427 mg) wasadded and the mixture cooled in an ice bath. Under a slow stream ofnitrogen solid sodium hydride-oil mixture (1.33 g, 60% NaH, 34.7 mmol)was added in small portions. The reaction mixture was stirred overnightwhile warming slowly to ambient temperature. The reaction was carefullypoured into a mixture of saturated ammonium chloride and ice andextracted three times with ethyl ether. The ether portions werecombined, washed with brine, dried (MgSO₄), filtered and the solventevaporated to yield a cloudy oil. The oil was purified by flashchromatography on a 120-gram silica gel column using a gradient of ethylacetate in hexane (1-30%) to give 93 as a clear oil (2.37 g, 4.72 mmol,41%). ¹H NMR (400 MHz, CDCl₃) δ 7.42 7.42 (m, 6H), 7.26 (m, 9H), 5.85(d, J=3.6 Hz, 1H), 4.50 (d, J=3.6 Hz, 1H), 4.343 (m, 1H), 3.86 (d, J=3.6Hz, 1H), 3.45 (m, 2H), 3.32 (m, 2H), 1.54 (m, 3H), 1.41 (m 2H), 1.33 (s,3H), 1.22 (m, 10H), 0.889 (t, J=6.8 Hz, 3H)

Step 2: Acetic acid 1,2,5-tri-O-acetyl-3-octyloxy-D-xylofuranose α and βmixture (94)

The trityl compound 93 (4.12 g, 7.57 mmol) was dissolved in a mixture ofacetic acid (35 ml) and acetic anhydride (15 ml) and cooled in a coolwater bath. A mixture of sulfuric acid in acetic anhydride and aceticacid was added (0.5 ml sulfuric acid, 2 ml acetic acid, 2 ml aceticanhydride, pre-cooled in an ice bath before addition) and the mixturestirred at ambient temperature over night. The reaction was poured onto400 g of ice water and extracted three times with ethyl acetate. Theorganic portions were combined, washed with saturated sodiumbicarbonate, dried (MgSO₄), filtered and evaporated to yield asemisolid. This was purified using flash chromatography on a 120 gsilica gel column eluting with a gradient of ethyl acetate in hexane(5-60%) to give 94 (1.12 grams, 2.88 mmol, 38%) as an oily mixture ofanomers. ¹H NMR (400 MHz, CDCl₃) δ 6.39 (d, J=3.6 Hz), 6.1 (s), 5.19 (m)4.46-4.52 (m), 4.31-4.43 (m), 4.11-4.25 (m), 3.93 (m), 3.45-3.68 (m),3.4-3.46 (m), 2.07-2.1 (m), 1.540 (m), 1.27 (m), 0.882 (t, J=6.8 Hz)

Step 3: Preparation of5-Amino-3-(3′-octyloxy-β-D-xylofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one(92)

In a manner similar to Example 23, step 1, using Acetic acid1,2,5-tri-O-acetyl-3-octyloxy-D-xylofuranose α and β mixture (94),afforded 80 mg (11%) of 92 as a fluffy white solid: ¹H NMR (400 MHzDMSO-d₆) δ 0.84-0.88 (m, 3H), 1.23-1.30 (m, 10H), 1.49-1.52 (m, 2H),2.00 (s, 3H), 2.02 (s, 3H), 3.41-3.44 (m, 1H), 3.57-3.59 (m, 1H),4.16-4.21 (m, 1H), 4.30-4.37 (m, 3H), 5.87 (d, J=5.6, 1H), 6.12 (dd,J=4.4, 1.2 Hz, 1H), 6.85 (br s, 2H), 8.35 (s, 1H); MS (ESI) [(M+H)⁺]found 497.40. Elemental analysis for (C₂₂H₃₂N₄O₇S): C, 53.21; H, 6.50;N, 11.28. Found C, 53.52; H, 6.49; N, 11.21.

Example 32 Preparation of 5-Amino-3-(3′-(R)-(2-methoxy-ethoxy),2′,5′-di-O-acetyl-β-D-xylofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one(95)

The required sugar, acetic acid1,2,5-tri-O-acetyl-3-(2-methoxy-ethoxy)-D-xylofuranose α and β mixture(98) as follows:

Step 1:1,2-O-isopropylidene-3-(2-methoxy-ethoxy)-5-O-trityl-D-xylofuranose (96)

Trityl alcohol 69 (5 grams, 11.57 mmol) was mixed with2-bromoethylmethyl ether (2.17 ml, 23.14 mmol) in THF (40 ml).Tetrabutylammonium iodide (427 mg) was added and the mixture cooled inan ice bath. Under a slow stream of nitrogen solid sodium hydride-oilmixture (1.33 g, 60% NaH, 34.7 mmol) was added in small portions. Thereaction was stirred overnight while warming slowly to ambienttemperature. The reaction was carefully poured into a mixture ofsaturated ammonium chloride and ice and extracted three times with ethylether. The ether portions were combined, washed with brine, dried(MgSO₄), filtered and the solvent evaporated to yield a cloudy oil. Theoil was purified by flash chromatography on a 120 g silica gel columnusing a gradient of ethyl acetate in hexane (3-30%). The ether product96 was isolated as a thick oil (4.54 g, 9.26 mmol, 80%). ¹H NMR (400MHz, CDCl₃) δ 7.42 (m, 6H), 7.26 (m, 9H), 5.86 (d, J=3.6 Hz, 1H), 4.54(d, J=4 Hz, 1H), 4.37 (m, 1H), 4.35 (m, 1H), 3.95 (d, J=2.8 Hz, 1H),3.64-3.68 (m, 1H), 3.47-3.52 (m, 2H) 3.29-3.33 (m, 2H), 3.24 (s, 3H)1.53 (s, 3H), 1.326 (s, 3H)

Step 2:1,2-O-isopropylidene-3-(2-methoxy-ethoxy)-5-O-acetyl-D-xylofuranose (97)

The trityl ether 96 (5.5 g, 11.22 mmol) was dissolved in aceticanhydride (30 ml) and acetyl bromide (2.0 ml, 22.4 mmol) was added.After one hour the reaction was filtered and the filtrate evaporated todryness. The residue was purified using flash chromatography on a 120 gsilica gel column using a gradient of ethyl acetate in hexane (10-100%)to yield 1.54 g (5.31 mmol, 47%) of acetate 97. ¹H NMR (400 MHz, CDCl₃)δ 5.925 (d, J=3.6 Hz, 1H), 4.58 (d, J=3.6 Hz, 1H), 4.38 (m, 2H), 4.23(m, 1H), 3.92 (d, J=3.6 Hz, 1H), 3.72 (m, 1H), 3.60 (m, 1H), 3.50 (m,2H), 3.35 (s, 3H), 2.08 (s, 3H), 1.49 (s, 3H), 1.32 (s, 3H).

Step 3: Acetic acid1,2,5-tri-O-acetyl-3-(2-methoxy-ethoxy)-D-xylofuranose α and β mixture(98)

The acetate 97 (1.71 grams, 5.89 mmol) was dissolved in a mixture ofacetic anhydride and acetic acid (1:4, 30 ml) and cooled in an ice bath.A solution of sulfuric acid in acetic acid (125 uL H₂SO₄ in 1.0 mlacetic anhydride) was added and the mixture maintained at −10 degreesovernight. The cold solution was poured onto 80 g of ice, let stand for20 minutes and then extracted three times with ethyl acetate. Theorganic portions were combined, washed with brine, dried (MgSO₄),filtered and the solvent removed to get 1.94 g of crude product. Thecrude product was purified using flash chromatography on a 120 g silicagel column eluting with a gradient of ethyl acetate in hexane (10-75%)to yield 760 mg (2.27 mmol, 38%) of 98 as a mixture of anomers. ¹H NMR(400 MHz, CDCl₃) δ 6.40 (d, J=4.4 Hz), 6.10 (s), 5.21 (m), 4.47-4.54(m), 4.33-4.45 (m), 4.16-4.27 (m), 4.03 (m), 3.72-3.85 (m), 3.6-3.7 (m),3.48-3.54 (m), 3.35-3.48 (m), 2.06-2.11 (m)

Step 4: Preparation of 5-Amino-3-(3′-(2-methoxy-ethoxy),2′,5′-di-O-acetyl-β-D-xylofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one(95)

In a manner similar to Example 23, step 1, using Acetic acid1,2,5-tri-O-acetyl-3-(2-methoxy-ethoxy)-D-xylofuranose α and β mixture(98), afforded 220 mg (42%) of 95 as a fluffy white solid: ¹H NMR (400MHz DMSO-d₆) δ 2.04 (d, J=8.4 Hz, 6H), 3.29 (s, 3H), 3.49-3.52 (m, 2H),3.58-3.63 (m, 1H), 3.76-3.81 (m, 1H), 4.19-4.24 (m, 1H), 4.36-4.43 (m,3H), 5.88 (d, J=6 Hz, 1H), 6.18 (dd, J=3.6, 2 Hz, 1H), 6.87 (br s, 2H),8.38 (s, 1H); MS (ESI) [(M+H)] found 443.31. Elemental analysis for(C₁₇H₂₂N₄O₈S.0.1H₂O.0.2EtOAc): C, 46.29; H, 5.19; N, 12.13. Found C,46.09; H, 5.25; N, 11.72.

Example 33 Preparation of5-Amino-3-(3′-Butoxy-α-D-xylofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one(99)

The required sugar acetic acid1,2,5-tri-O-acetyl-3-butoxy-D-xylofuranose α and β mixture (101) wasprepared as follows:

Step 1: Preparation of1,2-O-isopropylidene-3-butoxy-5-O-acetyl-D-xylofuranose (100)

Trityl alcohol 69 (5 grams, 11.57 mmol) was mixed with n-butyliodide(2.6 ml, 23.14 mmol) in THF (40 ml). Tetrabutylammonium iodide (427 mg)was added and the mixture cooled in an ice bath. Under a slow stream ofnitrogen solid sodium hydride-oil mixture (1.33 g, 60% NaH, 34.7 mmol)was added in small portions. The reaction was stirred overnight whilewarming slowly to ambient temperature. The reaction was carefully pouredinto a mixture of saturated ammonium chloride and ice and extractedthree times with ethyl ether. The ether portions were combined, washedwith brine, dried (MgSO₄), filtered and the solvent evaporated to yielda cloudy oil. The oil was purified by flash chromatography on a 120 gsilica gel column using a gradient of ethyl acetate in hexane (1-30%) togive 100 as a clear oil (2.32 g, 4.75 mmol, 41%). ¹H NMR (400 MHz,CDCl₃) δ 7.42 (m, 6H), 7.26 (m, 9H), 5.86 (d, J=3.6 Hz, 1H), 4.51 (d,J=3.6 Hz, 1H), 4.35 (m, 1H), 3.86 (d, J=3.6 Hz, 1H), 3.46 (m, 2H), 3.29(m, 2H), 1.54 (m, 3H), 1.38 (m, 2H), 1.33 (s, 3H), 1.23 (m, 2H), 0.83(t, J=7.6 Hz, 3H)

Step 2: Preparation Acetic acid1,2,5-tri-O-acetyl-3-butoxy-D-xylofuranose α and β mixture (101)

The trityl compound 100 (2.32 g, 4.75 mmol) was dissolved in 5% aceticanhydride in acetic acid (50 ml), cooled in a cool water bath and 0.02ml of sulfuric acid was added and the mixture stirred overnight atambient temperature. The reaction mixture was poured onto 150 grams ofice and extracted three times with methylene chloride. The organicportions were dried (MgSO₄), filtered and taken to dryness with tolueneto give 3.19 g of a semi-solid. This was purified using flashchromatography on a 50 g silica gel column eluting with a gradient ofethyl acetate in hexane (5-75%) to give 101 (0.760 g, 2.29 mmol, 48%) asan oily mixture of anomers. ¹H NMR (400 MHz, CDCl₃) δ 6.38 (d, J=3.6Hz), 6.11 (s), 5.2 (m), 4.50 (m), 4.31-4.42 (m), 4.13-4.25 (m), 3.93 (d,J=3.6 Hz), 3.5-3.7 (m), 3.4-3.47 (m), 2.06-2.15 (m), 1.51-1.55 (m),1.3-1.4 (m), 0.89-0.94 (m)

Step 3: Preparation of5-Amino-3-(3)-Butoxy-2′,5′-di-O-acetyl-α-D-xylofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one(102)

In a manner similar to Example 23, step 1, using Acetic acid1,2,5-tri-O-acetyl-3-butoxy-D-xylofuranose α and β mixture (101),afforded 40 mg (8%) of 102 as a white solid. Taken crude on to step 2.

Step 2: Preparation of5-Amino-3-(3′-Butoxy-α-D-xylofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one(99)

In a manner similar to Example 23, step 2, 102 afforded 5 mg (15%) of 99as a white solid: ¹H NMR (400 MHz, (CDCl₃) δ 0.84 (t, J=7.2 Hz, 3H),1.27-1.32 (m, 2H), 1.51-1.54 (m, 2H), 3.20 (t, J=9.2 Hz, 1H), 3.35 (t,J=10.8 Hz, 1H), 3.72-3.84 (m, 3H), 3.97-4.01 (m, 1H), 4.74 (t, J=9.2 Hz,1H), 5.17 (br s, 2H), 5.38 (d, J=9.2 Hz, 1H), 7.96 (s, 1H); MS (ESI)[(M+H)⁺] found 356.80.

Example 34 Preparation of 5-Amino-3-(3′-methyl,2′,3′,5′-tri-O-acetyl-β-D-xylofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one(103)

The preparation of the required sugar, 1,2,3,5tetra-O-acetyl-3(S)-methyl D-xylofuranose α and β mixture (105), wasprepared as follows:

Step 1: 1,2,3,5 tetra-O-acetyl-3-methyl D-xylofuranose α and β mixture(105)

The diol 104 [prepared as described by Lu and Just; Tetrahedron Letters41 (2000) 9223-9227] (1.69 g, 8.28 mmol), was dissolved in methylenechloride (25 ml) and pyridine (4.7 ml) was added. Acetic anhydride (3.9ml, 41 mmol) was added along with DMAP (50 mg) and this mixture wasstirred overnight at ambient temperature. The reaction was diluted withmethylene chloride and washed with saturated ammonium chloride. Theaqueous phase was extracted twice more with methylene chloride, theorganic portions combined, dried (MgSO₄), filtered and evaporated togive a colorless oil. The oil was dissolved in 5% acetic anhydride inacetic acid (68 ml) and sulfuric acid (0.02 ml) was added and this wasstirred overnight at ambient temperature. The reaction was poured onto150 g of ice, extracted three times with methylene chloride, the organicphases combined, washed twice with saturated sodium bicarbonate, dried(MgSO₄), filtered and evaporated to get 2.84 g of an oil. The residuewas purified using flash chromatography on a 120 g silica gel columnusing a gradient of ethyl acetate in hexane (5-75%) to yield 1.2 g (3.61mmol, 44%) of 105 as a clear oil whose spectra are consistent with amixture of anomers. ¹H NMR (400 MHz, CDCl₃) δ 6.41 (d, J=4.8 Hz), 6.03(d, J=1.2 Hz), 5.75 (d, J=0.8 Hz), 5.49 (d, J=5.2 Hz), 4.37-4.45 (m),4.2-4.29 (m), 2.03-2.135 (m), 1.637 S), 1.624 (s)

Step 2: Preparation of 5-Amino-3-(3′-methyl,2′,3′,5′-tri-O-acetyl-β-D-xylofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one(103)

In a manner similar to Example 23, step 1, using 1,2,3,5tetra-O-acetyl-3-methyl D-xylofuranose α and β mixture (105), afforded170 mg (28%) of 103 as a white solid: ¹H NMR (400 MHz DMSO-d₆) δ 1.57(s, 3H), 2.03 (s, 6H), 2.07 (s, 3H), 4.04 (dd, J=8.0, 2.8 Hz, 1H), 4.24(m, 1H), 4.41 (dd, J=12.0, 2.8 Hz, 1H), 5.73 (d, J=4.8 Hz, 1H), 6.24 (d,J=4.4 Hz, 1H), 6.89 (br s, 2H), 8.36 (s, 1H); MS (ESI) [(M+H)⁺] found441.08. Elemental analysis for (C₁₇H₂₀N₄O₈S.0.3H₂O): C, 45.80; H, 4.66;N, 12.57. Found C, 45.84; H, 4.50; N, 12.47.

Example 35 Preparation of 5-Amino-3-(5′-(1,2-diacetoxy-ethyl),2′,3′-di-O-acetyl-β-D-glucofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one(106)

The required sugar, penta-O-acetylglucofuranose (108) was prepared asdescribed below.

Step 1: Penta-O-acetylglucofuranose (108)

1,2-O-Isopropylidine-α-D-glucofranose (107) (5 g, 22.7 mmol) wasdissolved in acetic acid (180 ml) and acetic anhydride (21.5 ml), cooledin a cool water bath and sulfuric acid (0.02 ml, 98%) was added and themixture stirred 24 hours at ambient temperature. The mixture was pouredonto 500 g of ice, water was added and this was extracted four timeswith methylene chloride. The organic portions were combined, washedtwice with saturated sodium bicarbonate, dried (MgSO₄), and filtered toyield an oily residue. This was purified on a 120 g silica gel columnusing a gradient of ethyl acetate in hexane (20-100%) to give 5.33 g(13.66 mmol, 60%) of 18 as an oil whose spectra are consistent with amixture of anomers. ¹H NMR (400 MHz, CDCl₃) δ 6.41 (d, J=3.6 Hz), 6.12(s), 5.58 (m), 5.41 (d, J=3.6 Hz), 5.20-5.37 (m), 4.56-4.62 (m),4.02-4.18 (m), 2.00-2.13 (m).

Step 2: Preparation of 5-Amino-3-(5′-(1,2-diacetoxy-ethyl),2′,3′-di-O-acetyl-β-D-glucofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one(106)

In a manner similar to Example 23, step 1, usingpenta-O-acetylglucofuranose (108), afforded 80 mg (9%) of 106 as a whitesolid: ¹H NMR (400 MHz CDCl₃) δ 2.07 (s, 6H), 2.09 (s, 3H), 2.14 (s,3H), 4.03-4.07 (m, 1H), 4.47 (dd, J=6.4, 2 Hz, 1H), 4.67 (dd, J=12.4,2.0 Hz, 1H), 5.31 (br s, 2H), 5.58-5.6 (m, 1H), 5.68 (m 1H), 5.97 (d,J=5.6 Hz, 1H), 6.10 (dd, J=3.6, 2 Hz, 1H), 8.16 (s, 1H); MS (ESI[(M+H)⁺] found 499.40. Elemental analysis for (C₁₉H₂₂N₄O₁₀S.0.1 IPA): C,45.95; H, 4.56; N, 11.11. Found C, 45.92; H, 4.76; N, 10.80.

Example 36 Preparation of 5-Amino-3-(3′-acetoxymethyl-,2′,3′,5′-tri-O-acetyl-β-D-xylofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one(109)

The required sugar, tetra-O-acetyl-3-acetoxymethyl-D-xylofuranos α and βmixture (113) was prepared as follows:

Step 1:1,2-O-isopropylidfene-3(S)-(hydroxy-hydroxymethyl)-D-xylofuranose (111)

The epoxide 110 [prepared as described by Lu and Just; TetrahedronLetters 41 (2000) 9223-9227] (1.68 g, 8.3 mmol) was dissolved in dioxane(9 ml) and 1.0M NaOH was added (16.6 ml, 16.6 mmol) and the reactionheated to 50 degrees for 30 minutes. The reaction was cooled to ambienttemperature, 16.6 ml of 1.0 M HCl and 100 ml of absolute ethanol wereadded, stirred 5 minutes and the mixture evaporated under vacuum to givea solid. The solid was suspended in 200 ml of CH₂Cl₂ and sonicated togive a very fine suspension of solid. This was dried (MgSO₄), filteredthrough Celite and evaporated to give 111 as a thick oil (1.81 grams,8.22 mmol, 99%). ¹H NMR (400 MHz, CDCl₃) δ 5.94 (d, J=3.6 Hz, 1H), 4.4(d, J=4 Hz, 2H), 4.03 (m, 2H), 3.83 (d, J=11.6, 1H), 3.78 (d, J=12 Hz,1H), 2.66 (bs, 2H, OH), 1.7 (bs, 1H, OH), 1.52 (s, 3H), 1.33 (s, 3H)

Step 2:1,2-O-isopropylidene-3-(acetoxy-methylacetoxy)-5-O-acetyl-D-xylofuranose(112)

The triol 111 (1.81 g, 8.22 mmol) was dissolved in pyridine (30 ml),acetic anhydride (7.75 ml, 82 mmol) was added followed by DMAP (50 mg)and the mixture stirred for 72 hours. The volatiles were evaporatedunder vacuum and the residue partioned between methylene chloride andsaturated ammonium chloride. The aqueous phase was extracted twicemethylene chloride and the organic portions combined, dried (MgSO₄),filtered and the solvent evaporated to get 2.83 g of an oil. The residuewas purified using flash chromatography on a 120 g silica gel columnusing a gradient of ethyl acetate in hexane (10-80%) to yield 1.82 g(5.26 mmol, 64%) of 112 as a clear oil. %). ¹H NMR (400 MHz, CDCl₃) δ5.92 (d, J=3.6 Hz, 1H), 5.02 (m, 2H), 4.51-4.58 (m, 2H), 4.35 (dd, J=8.4Hz, J=2.8 Hz, 1H), 4.16-4.22 (m, 1H), 2.10 (s, 3H), 2.09 (s, 3H), 2.08(s, 3H), 1.53 (s, 3H), 1.32 (s, 3H)

Step 3: Tetra-O-acetyl-3-(acetoxymethyl)-D-xylofuranose, α and β mixture(113)

The triacetate 112 (1.74 g, 5.01 mmol) was dissolved in acetic acid (45ml), acetic anhydride was added (2.37 ml, 25 mmol) followed by sulfuricacid in acetic acid (0.5 ml of a 1.0M solution, 0.5 mmol) and thismixture was stirred overnight at ambient temperature. The reaction wasdiluted with methylene chloride (70 ml) and washed with water. The waterlayer was extracted twice with methylene chloride. The organic portionswere combined, transferred to a large beaker and saturated sodiumbicarbonate was added. To this was added solid sodium bicarbonate untilno more bubbling is observed. Separate the organic phase, extract theaqueous phase with methylene chloride, combine the organic phases anddry (MgSO₄), filter and evaporate to get an oil that was further takento dryness with toluene to yield 113 as a clear oil (1.91 g, 3.89 mmol,97%) whose NMR is consistent with a mixture of anomers. ¹H NMR (400 MHz,CDCl₃) δ 6.42 (d, J=4.4 Hz), 6.05 (d, J=1.6 Hz), 5.79 (d, J=1.6 Hz), 5.5(d, J=4.4 Hz), 4.89-4.93 (m), 4.12-4.58 (m), 2.04-2.2 (many singlets).

Step 4: Preparation of 5-Amino-3-(3′-acetoxymethyl-,2′,3′,5′-tri-O-acetyl-β-D-xylofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one(109)

In a manner similar to Example 23, step 1, usingtetra-O-acetyl-3-acetoxymethyl-D-xylofuranos α and β mixture (113)afforded 272 mg (37%) of 109 as a white solid: ¹H NMR (400 MHz (DMSO-d₆)δ 2.03 (s, 3H), 2.05 (s, 3H), 2.07 (s, 3H), 2.09 (s, 3H), 4.20 (m, 1H),4.44-4.57 (m, 3H), 4.79 (d, J=12.4 Hz, 1H), 5.84 (d, J=5.6 Hz, 1H), 6.38(d, J=6 Hz, 1H), 6.87 (br s, 2H), 8.37 (s, 1H); MS (ESI) [(M+H)⁺] found499.12. Elemental analysis for (C₁₉H₂₂N₄O₁₀S.0.1H₂O): C, 45.61; H, 4.47;N, 11.20. Found C, 45.93; H, 4.44; N, 10.83.

Example 37 Preparation of5-Amino-3-(2′,3′,5′-tri-O-acetyl-β-D-xylofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one(114)

Step 1: Preparation of5-Amino-3-(2′,3′,5′-tri-O-acetyl-β-D-xylofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one(114)

In a manner similar to Example 23, step 1, using commercially availabletetra-O-acetylxylofuranose, afforded 110 mg (14%) of 114 as a whitesolid: ¹H NMR (400 MHz (CDCl₃) δ 2.08 (s, 3H), 2.10 (s, 3H), 2.18 (s,3H), 4.42-4.45 (m, 2H), 4.52-4.56 (m, 1H), 5.13 (br s, 2H), 5.49 (dd,J=3.6, 2.4 Hz, 1H), 6.00 (d, J=5.2 Hz, 1H), 6.22 (dd, J=4, 1.6 Hz, 1H),8.15 (s, 1H); MS (ESI) [(M+H)⁺] found 426.93. Elemental analysis for(C₁₆H₁₈N₄O₈S): C, 45.07; H, 4.25; N, 13.14. Found C, 44.86; H, 4.17; N,13.05.

Example 385-Amino-3-(3′-C-methyl-β-D-ribofuranosyl)thiazolo[4,5-d]pyrimidine-2,7(3H,6H)-dione(117)

Step 1) Preparation of5-Amino-3-(3′-O-acetyl-2′,5′-di-O-benzoyl-3′-C-methyl-β-D-ribofuranosyl)thiazolo[4,5-d]pyrmidine-2,7(3H,6H)-dione(116)

5-Amino-3H,6H-thiazolo[4,5-d]pyrimidine (116 mg, 0.631 mmol),3′-C-methyl-ribfuranose 11 (272 mg, 0.598 mmol) [prepared according tothe method of Giradet, et. al. J. Med. Chem. 2000, 43, 3704-3713], BSA(0.462 ml, 1.89 mmol) and acetonitrile (5 mL) were mixed vigorously atambient temperature for 40 min. Once a homogeneous solution wasobtained, the reaction was charged with TMSOTf (0.171 mL, 210 mg). Thenthe reaction was heated to 60° C. After 1 h the solvent was removed byrotary evaporation. The resultant solid was dissolved in ethyl acetate(10 mL) and extracted with saturated sodium bicarbonate (2×5 mL). Theaqueous phase was then back extracted with ethyl acetate (5 mL) and theorganic layers were combined. A solid impurity promptly precipitated outof the organic phase, this was filtered off and discarded. The organicphase was concentrated and the resultant solid then triturated in ether(5 mL) yielding 167 mg (45%) of tan solid: ¹H NMR (400 MHz, d₆-DMSO) δ12.2 (br s, 1H), 8.01 (m, 4H), 7.83 (m, 2H), 7.53 (m, 4H), 7.28 (br s,2H), 6.4 (m, 1H), 6.02 (m, 1H), 4.82-4.67 (m, 2H), 3.38 (s, 1H), 2.01(s, 3H), 1.98 (s, 3H); [M+H]⁺ m/z 581.

Step 2) Preparation of5-Amino-3-(3′-C-methyl-β-D-ribofuranosyl)thiazolo[4,5-d]pyrimidine-2,7(3H,6H)-dione(117)

Nucleoside triester 116 (100 mg, 0.172 mmol) was dissolved in methanol(5 mL) and K₂CO₃ (28.6 mg, 0.207 mmol) was added. The reactionprogressed for 16 h. The reaction was neutralized with acetic acid (24.8mg, 0.412 mmol). Then the solvent removed by rotary vacuum and the solidsubmitted to HPLC purification (MeCN—H₂O) yielding 42 mg (74%) of whitesolid: ¹H NMR (400 MHz, d₆-DMSO) δ 11.20 (s, 1H), 6.89 (br s, 2H), 5.80(d, J=8.0, 1H), 5.36 (d, J=6.0, 1H), 4.77 (t, J=8.0, 1H), 4.62 (s, 1H),4.48 (m, 1H), 3.75 (m, 1H), 3.58-3.44 (m, 2H), 1.19 (s, 3H); Analysiscalc'd for C₁₄H₁₄N₄O₆S.0.125H₂O.0.125HCO₂H: C, 39.33; H, 4.34; N, 16.43;S, 9.40. Found: C, 39.77; H, 4.81; N, 15.02; S, 9.69; [M+H]⁺ m/z 331.

Example 395-Amino-3-(2′-C-methyl-β-D-ribofuranosyl)-3H,6H-thiazolo[4,5-d]pyrimidine-2,7-dione(120)

Step 1) Preparation of5-Amino-3-(2′,3′,5′-tri-O-benzoyl-2′-C-methyl-β-D-ribofuranosyl)-3H,6H-thiazolo[4,5-d]pyrimidine-2,7-dione(119)

To a suspension of heterocycle 4 (268 mg, 1.44 mmol) in anhydrous MeCN(8 mL) at rt was added BSA (971 uL, 3.93 mmol). The resultant mixturewas heated to 80° C. for 2.5 h whereupon 2-C-methyl-β-D-ribofuranose 118[prepared according to Wolfe et al. J. Org. Chem. 1997, 62, 1754-1759](760 mg, 1.31 mmol) was added as a solution in MeCN (6 mL). To thismixture was added SnCl₄ (276 uL, 2.35 mmol), and stirring at 80° C. wascontinued for an additional 1.5 h. TLC analysis with 10% MeOH—CHCl₃indicated that the reaction was complete. The mixture was cooled to rt,diluted with EtOAc (150 mL), and partitioned with a 1:1 mixture (100 mL)of brine-NaHCO₃. The aqueous phase was further extracted with EtOAc (50mL), and the combined organic phases were dried over Na₂SO₄, filteredand concentrated. The residue was submitted to HPLC (SiO₂, 0-4%MeOH-DCM) to afford 353 mg (42%) of a white solid: ¹H (400 MHz, DMSO-d₆)δ 11.3 (br s, 1H), 7.93-8.08 (br m, 3H), 7.85-7.87 (m, 2H), 7.33-7.66(m, 10H), 6.97 (br s, 2H), 6.64 (s, 1H), 6.16-6.26 (br m, 1H), 4.56-4.79(br m, 3H), 1.79 (s, 3H); M⁺ m/z 642.

Step 2) Preparation of5-Amino-3-(2′-C-methyl-β-D-ribofuranosyl)-3H,6H-thiazolo[4,5-d]pyrimidine-2,7-dione(120)

A mixture of nucleoside triester 119 (209 mg, 0.325 mmol) and MeOH (10mL) saturated with NH₃ (g) at −30° C. was stirred in a sealed tube for48 h. The mixture was warmed to rt, depressurized, concentrated thensubmitted to HPLC purification (MeCN—H₂O), affording 36 mg (34%) of thetitle compound as a white solid after lyophilization: ¹H (400 MHz,DMSO-d₆) δ 11.18 (s, 1H), 6.92 (br s, 2H), 5.95 (s, 1H), 5.18-5.28 (brm, 1H), 4.75 (br s, 1H), 4.53 (dd, J=11.3, 5.46, 1H), 3.95 (br s, 1H),3.73-3.78 (m, 1H), 3.29 (br s, 2H), 1.04 (s, 3H); [M+H]⁺ m/z 331.

Example 405-Amino-3-(3′-deoxy-β-D-ribofuranosyl)-3H,6H-thiazolo[4,5-d]pyrimidine-2,7-dione(122)

Step 1) Preparation of5-Amino-3-(2′,5′-di-O-acetyl-3′-deoxy-β-D-ribofuranosyl)-3H,6H-thiazolo[4,5-d]pyrimidine-2,7-dione(121)

To a suspension of heterocycle 6 (4.60 g, 25.0 mmol) anddeoxyribofuranose 11B (5.42 g, 20.8 mmol) in MeCN (83 mL) at rt wasadded BSA (15.3 mL, 62.5 mmol). The resultant mixture was immersed intoa 40° C. oil bath for 1.5 h, and TMSOTf (5.65 mL, 31.2 mmol) was addeddropwise. The thick reaction mixture was immersed into an 80° C. oilbath and stirred for 2.5 h whereupon it was concentrated via rotaryevaporation to a residue that was partitioned between EtOAc (300 mL) andpH 7 buffer (100 mL). The organic phase was dried over Na₂SO₄ andconcentrated to a residue that was triturated with EtOAc and thenfiltered to yield 2.31 g (29%) of fine white solid. The filtrate wasconcentrated to a residue that was submitted to flash chromatography(SiO₂, 0-6% MeOH-DCM) to afford 1.12 g (14%) of very fine pale yellowsolid. Altogether, there was a 43% combined yield of nucleoside 121: ¹H(400 MHz, DMSO-d₆) δ 11.23 (s, 1H), 6.95 (br s, 2H), 5.79 (d, J=2.0,1H), 5.59 (d, J=7.2, 1H), 4.28-4.34 (m, 1H), 4.22 (dd, J=3.2, 12.0, 1H),3.99 (dd, J=6.4, 11.6, 1H), 2.56-2.65 (m, 1H), 2.04 (s, 3H), 1.98 (s,3H), 1.97-2.04 (m, 1H); [M+H]⁺ m/z 384.8. Analysis cal'd for:C₁₄H₁₆N₄O₇S.0.5H₂O: C, 42.74; H, 4.36; N, 14.24; S, 8.15. Found: C,42.72; H, 4.22; N, 14.15; S, 8.19.

Step 2) Preparation of5-Amino-3-(3′-deoxy-β-D-ribofuranosyl)-3H,6H-thiazolo[4,5-d]pyrimidine-2,7-dione(122)

To a suspension of nucleoside diester 121 (1.91 g, 4.97 mmol) in MeOH(50 mL) at rt was added K₂CO₃ (820 mg, 5.97 mmol). The reaction mixturewas stirred for 18 h, then quenched with HOAc (0.68 mL, 12 mmol),stirred 30 min, and finally concentrated via rotary evaporation. Theresidue was azeotroped with toluene (3×50 mL), and then triturated withwater (250 mL). The solid material was filtered, washed with water(2×250 mL), air dried, triturated with ether (250 mL), and filtered toprovide 1.17 g (62%) of nucleoside 122 as an off-white solid: ¹H (400MHz, DMSO-d₆) δ 11.26 (br s, 1H), 6.93 (br s, 1H) 7.14 (d, J=2.4, 1H),5.39 (d, J=4.4, 1H), 4.74-4.79 (m, 1H), 4.65 (t, J=5.6, 1H), 4.09-4.16(m, 1H), 3.41-3.43 (m, 2H), 2.23-2.30 (m, 1H), 1.78 (ddd, J=2.4, 6.4,8.4, 1H) 1.76-1.81 (m, 1H); [M+H]⁺ @ m/z 301.5. Analysis cal'd for:C₁₀H₁₂N₄O₅S.1.25H₂O: C, 37.20; H, 4.53; N, 17.36; S, 9.93. Found: C,37.06; H, 4.27; N, 17.14; S, 9.84.

Example 415-Amino-3-(2′-deoxy-β-D-ribofuranosyl)-3H,6H-thiazolo[4,5-d]pyrimidine-2,7-dione(130)

Step 1) Preparation of5-N-Acetyl-amino-3-(β-D-ribofuranosyl)-3H,6H-thiazolo[4,5-d]pyrimidine-2,7-dione

To a suspension of isatoribine tetraacetate 123 [CAS #533897-42-6,prepared according to Webber et. al. U.S. Pat. No. 6,924,271] (12.3 g,25.4 mmol) in MeOH (180 mL) was added concentrated NH₄OH (180 mL). Theresultant mixture was stirred 1 h whereupon it was concentrated andsubmitted to flash chromatography (SiO₂, 15-30% IPA-CHCl₃) to afford2.50 g (27%) of acetamide 124 as a white solid: [M+H]⁺ m/z 359.

Step 2) Preparation of5-N-Acetyl-amino-3-(5′-O-tert-butyldimethylsilyl-β-D-ribofuranosyl)-3H,6H-thiazolo[4,5-d]pyrimidine-2,7-dione

To a solution of triol 124 (2.48 g, 6.93 mmol) in DMF (15 mL) was addedsequentially imidazole (943 mg, 13.9 mmol) and TBSCl (1.04 g, 6.93 mmol)at rt. The resultant mixture was stirred 1 h, then diluted with EtOAc(300 mL) and extracted with water (2×100 mL) then brine (100 mL). Theorganic phase was dried over Na₂SO₄, concentrated and triturated withether to afford 2.18 g (67%) of siloxane 125 as an off-white solid: ¹H(400 MHz, DMSO-d₆) δ 12.16 (br s, 1H), 11.81 (br s, 1H), 5.81 (d,J=4.77, 1H), 5.34 (d, J=5.13, 1H), 5.03 (d, J=5.50, 1H), 4.79 (dd,J=10.3, 5.13, 1H), 4.13 (dd, J=10.6, 5.5, 1H), 3.71-3.78 (m, 2H),3.40-3.64 (m, 1H), 2.18 (s, 3H), 0.84 (s, 9H), 0.00 (s, 6H); [M+H]⁺ m/z473.

Step 3) Preparation of5-N-Acetyl-amino-3-(5′-O-tert-butyldimethylsilyl-2′,3′-thioxo-β-D-ribofuranosyl)-3H,6H-thiazolo[4,5-d]pyrimidine-2,7-dione

To a solution of diol 125 (1.00 g, 2.12 mmol) in MeCN (50 mL) was addedTCDI (754 mg, 4.23 mmol) at rt. The resultant mixture was stirred for 18h whereupon it was concentrated, submitted to flash chromatography(SiO₂, 40% EtOAc-CHCl₃), and triturated with ether to afford 730 mg(67%) of a white solid: ¹H (400 MHz, DMSO-d₆) δ 12.18 (br s, 1H), 11.75(br s, 1H), 6.21-6.24 (m, 2H), 5.79 (br s, 1H), 4.35 (br s, 1H), 3.72(br d, J=6.6, 2H), 2.21 (s, 3H), 0.84 (s, 9H), 0.01 (s, 6H); [M+H]⁺ m/z515.

Step 4) Preparation of5-N-Acetyl-amino-3-(5′-O-tert-butyldimethylsilyl-2′-deoxy-β-D-ribofuranosyl)-3H,6H-thiazolo[4,5-d]pyrimidine-2,7-dione

To a suspension of thiocarbonate 126 (712 mg, 1.38 mmol) and Bu₃SnH(2.66 mL, 10.0 mmol) in anhydrous toluene (140 mL) was added AIBN (30mg, 0.18 mmol) at rt. The mixture was immersed into a 130° C. oil bathfor 15 min then removed, cooled, concentrated and submitted to flashchromatography (SiO₂, 80-100% EtOAc-CHCl₃) to afford 450 mg (71%) of amixture (2:1) of 2′-deoxy and 3′-deoxy regioisomers (major isomerreported): ¹H (400 MHz, DMSO-d₆) δ 12.12 (br s, 1H), 11.82 (br s, 1H),6.26 (t, J=7.0, 1H), 5.22 (d, J=4.0, 1H), 4.31-4.34 (m, 1H), 3.69-3.75(m, 2H), 3.57-3.62 (m, 1H), 2.93-2.99 (m, 1H), 2.18 (s, 3H), 2.00-2.18(m, 1H), 0.84 (s, 9H), 0.00 (s, 6H); [M+H]⁺ m/z 457.

Step 5) Preparation of5-N-Acetyl-amino-3-(2′-deoxy-β-D-ribofuranosyl)-3H,6H-thiazolo[4,5-d]pyrimidine-2,7-dione

To a suspension of the regioisomers (744 mg, 1.60 mmol) from Step 4(above) in MeCN (30 mL) at rt was added 48% aqueous HF (1.67 mL). Thereaction mixture was stirred 1 h whereupon it was concentrated to apurple residue that was submitted to flash chromatography (SiO₂, 1.5-15%MeOH-DCM), affording 503 mg (92%) of a mixture of regioisomers that wasfurther purified via HPLC (MeCN—H₂O) to provide 169 mg (31%) ofnucleoside 129 as a white solid after lyophilization: ¹H (400 MHz,DMSO-d₆) δ 12.14 (s, 1H), 11.85 (s, 1H), 6.26 (t, J=7.0, 1H), 4.30-4.32(m, 1H), 3.69-3.71 (m, 1H), 3.38-3.53 (m, 4H), 2.92-2.98 (m, 1H), 2.19(s, 3H), 1.97-2.03 (s, 1H); [M+H]⁺ m/z 343.

Step 6) Preparation of5-Amino-3-(2′-deoxy-β-D-ribofuranosyl)-3H,6H-thiazolo[4,5-d]pyrimidine-2,7-dione

To a solution of acetamide 129 (169 mg, 0.494 mmol) in MeOH (10 mL) wasadded K₂CO₃ (158 mg, 1.14 mmol) at rt. The resulting mixture was stirredfor 8 h whereupon it was quenched with HOAc (137 uL, 2.40 mmol),concentrated and submitted to HPLC (MeCN—H₂O) to afford 125 mg (84%) ofthe title compound 130 as a white solid after lyophilization: ¹H (400MHz, DMSO-d₆) δ 11.16 (s, 1H), 6.90 (br s, 2H), 6.22 (t, J=7.0, 1H),4.27-4.31 (m, 1H), 3.67-3.71 (m, 1H), 3.52 (dd, J=11.3, 5.5, 1H), 3.40(dd, J=11.7, 6.2, 1H), 2.86-2.93 (m, 1H), 1.97 (ddd, J=12.9, 7.0, 3.5,1H); [M+H]⁺ m/z 301. Analysis calc'd for C₁₀H₁₂N₄O₅S.H₂O: C, 37.73; H,4.43; N, 17.60; S, 10.07. Found: C, 38.13; H, 4.27; N, 17.40; S, 9.89.

Example 42 Preparation of5-Amino-3-(3′-deoxy-3′-methylidene-β-D-ribofuranosyl)-3H,6H-thiazolo[4,5-d]pyrimidine-2,7-dione(132)

Step 1: Preparation of5-Amino-3-(2′-O-acetyl-5′-O-benzoyl-3′-deoxy-3′-methylidene-β-D-ribofuranosyl)-3H,6H-thiazolo[4,5-d]pyrimidine-2,7-dione(131)

1,2-di-O-acetyl-5-O-benzoyl-3-deoxy-3-methylidene-α,β-D-ribofuranose(127) (132 mg, 0.39 mmol) [prepared according to the method of Girardetet al. J. Med. Chem. 2000, 43, 3704-3713] was dissolved in acetonitrile(5 mL) at ambient temperature.5-Amino-3H,6H-thiazolo[4,5-d]pyrimidine-2,7-dione (1) (73 mg, 0.39 mmol)was added, the mixture was then stirred for 0.5 h before it was heatedto 40° C. After 5 min at 40° C., BSA (0.29 mL, 1.18 mmol) was added andthe mixture was stirred for another 0.5 h. The mixture was then heatedto 80° C. TMSOTf (0.107 mL, 0.59 mmol) was added and the reaction wasstirred for 3-4 hours at 80° C. Upon completion, the reaction wasallowed to cool to room temperature and then quenched by a pH 7.0 buffer(1.0 M K₂HPO₄ and 1.0 M NaH₂PO₄, 2 ml). The mixture was extracted withCH₂Cl₂ (3×10 mL). The combined organic layers were washed with brine,dried with Na₂SO₄, and concentrated in vacuo. The crude product waspurified by column chromatography (SiO₂, 0-10% MeOH—CH₂Cl₂ to afford 23mg (13%) of 131 as a light yellow solid: ¹H NMR (400 MHz, CDCl₃) δ 9.85(s, 1H), 8.03 (d, J=8, 2H), 7.54 (m, 1H), 7.417 (t, J=8, 2H), 6.50 (s,2H), 6.07 (d, J=4.8, 1H), 5.73 (m, 1H), 5.37 (d, J=32, 2H), 4.83 (m,2H), 4.46 (m, 1H), 2.00 (s, 3H); [M+H]⁺459.3; Elemental analysis forC₂₀H₁₈N₄O₇S.0.7EtOAc: calc'd: C, 52.65; H, 4.57; N, 10.77; S, 6.16.found: C, 53.59; H, 4.57; N, 10.83; S, 6.17.

Step 2: Preparation of(3′S)-5-Amino-3-(3′-deoxy-3′-methylidene-β-D-ribofuranosyl)-3H,6H-thiazolo[4,5-d]pyrimidin-2,7-dione(132)

(3′S)-5-Amino-3-(3′-acetoxymethyl-2′,5′-di-O-acetyl-3′-deoxy-β-D-ribofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one131 (113 mg, 0.25 mmol) was dissolved in methanol (5 mL) at ambienttemperature. Potassium carbonate (38 mg, 0.27 mmol) was added and themixture was stirred at room temperature overnight. Upon completion,acetic acid was added (34 μL) and the mixture was stirred another 30minutes at room temperature. The mixture was concentrated, purified byHPLC, then triturated by EtOAc to afford 49 mg (64%) of 132 as a whitesolid: ¹H NMR (400 MHz, DMSO-d₆) δ 11.563 (s, 1H), 6.82 (s, 2H), 5.80(m, 1H), 5.62 (m, 1H), 5.16 (d, J=14.4, 2H), 4.51 (m, 1H), 3.53 (m, 2H),1.89 (s, 2H); [M+H]⁺ 313.07.

Example 43 Preparation of5-Amino-3-(2′,3′,5′-tri-hydroxy-β-D-xylofuranosyl)-3H,6H-thiazolo[4,5-d]pyrimidin-2-one(134)

Step 1: Preparation of5-Amino-3-(2′,3′,5′-tri-O-acetyl-β-D-xylofuranosyl)-3H,6H-thiazolo[4,5-d]pyrimidin-2-one(133)

In a manner similar to Example 42, Step 1, using commercially availabletetra-O-acetylxylofuranose, 740 mg of the title compound 133 wasgenerated in 20% yield as a white solid: ¹H NMR (400 MHz, d₆-DMSO) δ11.32 (s, 1H), 6.98 (br s, 2H), 6.09 (dd, J=8.6, 2.3 Hz, 1H), 5.76 (d,J=5.5 Hz, 1H), 5.38 (dd, J=8.6, 2.3 Hz, 1H), 4.14 (m, 1H), 4.26 (m, 1H),4.16 (m, 1H), 2.08 (s, 3H), 2.04 (s, 3H), 2.00 (s, 3H); [M+H]⁺ 442.8;Elemental Analysis for C₁₆H₁₈N₄O₉S.1.0H₂O: calc'd: C, 41.74; H, 4.38; N,12.17. found: C, 41.92; H, 4.23; N, 11.71.

Step 2: Preparation of5-Amino-3-(2′,3′,5′-tri-hydroxy-β-D-xylofuranosyl)-3H,6H-thiazolo[4,5-d]pyrimidin-2-one(134)

In a manner similar to Example 42, Step 2, 43 mg of the title compound134 was generated in 67% yield as a white solid: ¹H NMR (400 MHz,d₆-DMSO) δ 11.24 (br s, 1H), 6.86 (br s, 2H), 5.60 (d, J=4.68 Hz, 1H),5.57 (d, J=4.68 Hz, 1H), 5.04 (d, J=7.8 Hz, 1H), 4.64 (m, 1H), 4.41 (m,1H), 3.87 (m, 2H), 3.54 (m, 2H); [M+H]⁺ 316.9; Elemental Analysis forC₁₁H₁₄N₄O₅S.1.3H₂O: calc'd: C, 35.35; H, 4.33; N, 16.49. found: C,35.73; H, 4.21; N, 16.15.

Example 44 Preparation of5-Amino-3-(3′-(R)-octyloxy-β-D-xylofuranosyl)-3H,6H-thiazolo[4,5-d]pyrimidin-2-one(135)

The required sugar acetic acid1,2,5-tri-O-acetyl-3(S)-octyloxy-D-xylofuranose α and β mixture (94) wasprepared as reported in Example 31.

Step 1: Preparation of5-Amino-3-(3′-(R)-octyloxy-β-D-xylofuranosyl)-3H,6H-thiazolo[4,5-d]pyrimidin-2-one(135)

In a manner similar to Example 42, Step 1, 18.8 mg of the title compound135 was generated from acetic acid1,2,5-tri-O-acetyl-3(S)-octyloxy-D-xylofuranose α and β mixture (94) in2% yield as a white solid: ¹H NMR (400 MHz, d₆-DMSO) δ 11.21 (s, 1H),6.94 (br s, 2H), 6.12 (m, 1H), 5.74 (d, J=6.2 Hz, 1H), 4.30 (m, 3H),4.16 (m, 1H), 3.57 (m, 1H), 3.41 (m, 1H), 2.02 (d, J=8.6 Hz, 6H), 1.50(m, 2H), 1.26 (m, 10H), 0.86 (m, 3H); [M+H]⁺ 512.9; Elemental Analysisfor C₂₂H₃₂N₄O₈S: calc'd: C, 51.55; H, 6.29; N, 10.93. found: C, 51.47;H, 6.37; N, 10.77.

Example 45 Preparation of5-Amino-3-(3′-(R)-Methoxy-β-D-xylofuranosyl)-3H,6H-thiazolo[4,5-d]pyrimidin-2-one(137)

The required sugar, acetic acid1,2,5-tri-O-acetyl-3)-methoxy-D-xylofuranose α and β mixture (91) wasprepared as reported in Example 30.

Step 1: Preparation of5-Amino-3-(3′-(R)-Methoxy-β-D-xylofuranosyl)-3H,6H-thiazolo[4,5-d]pyrimidin-2-one(136)

In a manner similar to Example 42, Step 1, 230 mg of the title compound136 was generated from acetic acid1,2,5-tri-O-acetyl-3)-methoxy-D-xylofuranose α and β mixture (91) in 31%yield as a white solid: ¹H NMR (400 MHz, d₆-DMSO) δ 11.21 (s, 1H), 6.94(br s, 2H), 6.14 (m, 1H), 5.74 (d, J=6.2 Hz, 1H), 4.33 (m, 2H), 4.18 (m,2H), 3.35 (s, 3H), 3.30 (s, 3H), 2.04 (s, 3H), 2.01 (s, 3H); [M+H]⁺414.8; Elemental Analysis for C₁₅H₁₈N₄O₈S: calc'd: C, 43.48; H, 4.38; N,13.52. found: C, 43.12; H, 4.36; N, 13.17.

Step 2: Preparation of5-Amino-3-(3′-(R)-Methoxy-β-D-xylofuranosyl)-3H,6H-thiazolo[4,5-d]pyrimidin-2-one(137)

In a manner similar to Example 42, Step 2, 43 mg of the title compound137 was generated in 29% yield as a white solid: ¹H NMR (400 MHz,d₆-DMSO) δ 11.50 (br s, 1H), 6.98 (br s, 2H), 5.68 (d, J=5.5 Hz, 1H),5.60 (d, J=7.8 Hz, 1H), 5.10 (m, 1H), 4.48 (m, 1H), 4.08 (m, 1H), 3.84(m, 1H), 3.57 (m, 2H), 3.35 (s, 3H); [M+H]⁺ 330.9; Elemental Analysisfor C₁₁H₁₄N₄O₆S.0.7H₂O.0.1iPrOH: calc'd: C, 38.89; H, 4.68; N, 16.06.found: C, 38.78; H, 4.30; N, 15.84.

Example 46 Preparation of 5-Amino-3-(3′-(R)-(2-methoxy-ethoxy),2′,5′-di-hydroxy-β-D-xylofuranosyl)-3H,6H-thiazolo[4,5-d]pyrimidin-2-one(139)

The required sugar, acetic acid1,2,5-tri-O-acetyl-3-(2-methoxy-ethoxy)-D-xylofuranose α and β mixture(98) was prepared as reported in Example 32.

Step 1: Preparation of 5-Amino-3-(3′-(R)-(2-methoxy-ethoxy),2′,5′-di-O-acetyl-β-D-xylofuranosyl)-3H,6H-thiazolo[4,5-d]pyrimidin-2-one(138)

In a manner similar to Example 42, Step 1, 118 mg of the title compound138 was generated from Acetic acid1,2,5-tri-O-acetyl-3-(2-methoxy-ethoxy)-D-xylofuranose α and β mixture(98) in 21% yield as a white solid: ¹H NMR (400 MHz, d₆-DMSO) δ 11.23(s, 1H), 6.90 (br s, 2H), 6.13 (m, 1H), 5.74 (d, J=6.24 Hz, 1H), 4.33(m, 3H), 4.17 (m, 1H), 3.73 (m, 1H), 3.57 (m, 1H), 3.46 (m, 2H), 3.25(s, 3H), 2.03 (s, 3H), 2.01 (s, 3H); [M+H]⁺ 459.3; Elemental Analysisfor C₁₇H₂₂N₄O₉S.0.3H₂O.0.5EtOAc: calc'd: C, 44.93; H, 5.28; N, 11.03.found: C, 44.93; H, 5.01; N, 11.14.

Step 2: Preparation of 5-Amino-3-(3′-(R)-(2-methoxy-ethoxy),2′,5′-di-hydroxy-β-D-xylofuranosyl)-3H,6H-thiazolo[4,5-d]pyrimidin-2-one(139)

In a manner similar to Example 42, Step 2, 43 mg of the title compound139 was generated in 36% yield as a white solid: ¹H NMR (400 MHz,d₆-DMSO) δ 11.44 (br s, 1H), 6.97 (br s, 2H), 5.67 (d, J=5.46 Hz, 1H),5.60 (d, J=7.8 Hz, 1H), 5.10 (m, 1H), 4.39 (m, 1H), 4.08 (m, 1H), 3.98(m, 1H), 3.71 (m, 1H), 3.59 (m, 3H), 3.45 (m, 2H), 3.27 (s, 3H); [M+H]⁺374.9; Elemental Analysis for C₁₃H₁₈N₄O₇S.1.0H₂O.0.25EtOAc: calc'd: C,40.57; H, 5.35; N, 13.52. found: C, 40.81; H, 4.96; N, 13.40.

Example 47 Preparation of 5-Amino-3-(3′-(S)-methyl,2′,3′,5′-tri-hydroxy-β-D-xylofuranosyl)-3H,6H-thiazolo[4,5-d]pyrimidin-2-one(141)

The preparation of the required sugar, 1,2,3,5tetra-O-acetyl-3(S)-methyl D-xylofuranose α and β mixture (105), wasprepared as reported in Example 34.

Step 1: Preparation of 5-Amino-3-(3′-(S)-methyl,2′,3′,5′-tri-O-acetyl-β-D-xylofuranosyl)-3H,6H-thiazolo[4,5-d]pyrimidin-2-one(140)

In a manner similar to Example 42, Step 1, 110 mg of the title compound140 was generated from 1,2,3,5 tetra-O-acetyl-3(S)-methyl D-xylofuranoseα and β mixture (105) in 15% yield as a white solid: ¹H NMR (400 MHz,d₆-DMSO) δ 11.27 (br s, 1H), 6.98 (br s, 2H), 6.25 (d, J=4.68 Hz, 1H),5.77 (d, J=4.68 Hz, 1H), 4.40 (dd, J=9.4, 3.1 Hz, 1H), 4.22 (m, 1H),3.68 (dd, J=4.7, 3.1 Hz, 1H), 2.08 (s, 3H), 2.04 (s, 3H), 2.02 (s, 3H),1.56 (s, 3H); [M+H]⁺ 456.8; Elemental Analysis forC₁₇H₂₀N₄O₉S.0.5H₂O.0.2iPrOH: calc'd: C, 44.27; H, 4.77; N, 11.73. found:C, 44.45; H, 4.55; N, 11.62.

Step 2: Preparation of 5-Amino-3-(3′-(S)-methyl,2′,3′,5′-tri-hydroxy-β-D-xylofuranosyl)-3H,6H-thiazolo[4,5-d]pyrimidin-2-one(141)

In a manner similar to Example 42, Step 2, 31 mg of the title compound141 was generated in 54% yield as a white solid: ¹H NMR (400 MHz,d₆-DMSO) δ 11.36 (br s, 1H), 6.94 (br s, 2H), 5.70 (d, J=5.46 Hz, 1H),5.58 (d, J=4.7 Hz, 1H), 5.09 (br s, 1H), 4.48 (m, 2H), 3.59 (m, 3H),1.17 (s, 3H); [M+H]⁺ 330.9; Elemental Analysis for C₁₁H₁₄N₄O₆S.1.1H₂O:calc'd: C, 37.73; H, 4.66; N, 16.00. found: C, 37.66; H, 4.22; N, 15.60.

Example 48 Preparation of 5-Amino-3-(5′-(1,2-diacetoxy-ethyl),2′,3′-di-hydroxy-β-D-glucofuranosyl)-3H,6H-thiazolo[4,5-d]pyrimidin-2-one(143)

The required sugar, penta-O-acetylglucofuranose (108), was prepared asdescribed in Example 35.

Step 1: Preparation of 5-Amino-3-(5′-(1,2-diacetoxy-ethyl),2′,3′-di-O-acetyl-β-D-glucofuranosyl)-3H,6H-thiazolo[4,5-d]pyrimidin-2-one(142)

In a manner similar to Example 42, Step 1, 100 mg of the title compound142 was generated from penta-O-acetylglucofuranose (108) in 10% yield asa white solid: ¹H NMR (400 MHz, CDCl₃) δ 5.91 (m, 1H), 5.81 (br s, 2H),5.73 (d, J=6.2 Hz, 1H), 5.51 (m, 1H), 5.41 (m, 1H), 4.55 (dd, J=12.5,2.3 Hz, 1H), 4.29 (t, J=7.02 Hz, 1H), 3.90 (m, 1H), 1.96 (s, 3H), 1.93(s, 3H), 1.90 (s, 3H), 1.55 (br s, 1H); [M+H]⁺ 515.3; Elemental Analysisfor C₁₉H₂₂N₄O₁₁S.0.15MeOH: calc'd: C, 44.29; H, 4.39; N, 10.79. found:C, 44.69; H, 4.44; N, 10.41.

Step 2: Preparation of 5-Amino-3-(5′-(1,2-diacetoxy-ethyl),2′,3′-di-hydroxy-β-D-glucofuranosyl)-3H,6H-thiazolo[4,5-d]pyrimidin-2-one(143)

In a manner similar to Example 42, Step 2, 45 mg of the title compound143 was generated in 83% yield as a white solid: ¹H NMR (400 MHz,d₆-DMSO) δ 11.34 (br s, 1H), 6.96 (br s, 2H), 5.72 (d, J=4.7 Hz, 1H),5.63 (d, J=3.12 Hz, 1H), 5.11 (d, J=9.4 Hz, 1H), 4.56 (m, 2H), 4.39 (t,J=5.46 Hz, 1H), 3.96 (m, 1H), 3.74 (m, 2H), 3.51 (m, 1H), 3.36 (m, 1H);[M+H]⁺346.9; Elemental Analysis for C₁₁H₁₄N₄O₇S.1.0H₂O: calc'd: C,36.26; H, 4.43; N, 15.38. found: C, 36.20; H, 4.37; N, 15.01.

Example 49 Preparation of 5-Amino-3-(3′-(S)-acetoxymethyl-,2′,3′,5′-tri-O-acetyl-β-D-xylofuranosyl)-3H,6H-thiazolo[4,5-d]pyrimidin-2-one(144)

The required sugar, tetra-O-acetyl-3-acetoxymethyl-D-xylofuranos α and βmixture (113) was prepared as reported in Example 36.

Step 1: Preparation of 5-Amino-3-(3′-(S)-acetoxymethyl-,2′,3′,5′-tri-O-acetyl-β-D-xylofuranosyl)-3H,6H-thiazolo[4,5-d]pyrimidin-2-one(144)

In a manner similar to Example 42, Step 1, 80 mg of the title compound144 was generated from tetra-O-acetyl-3-acetoxymethyl-D-xylofuranos αand β mixture (113) in 13% yield as a white solid: ¹H NMR (400 MHz,d₆-DMSO) δ 11.23 (s, 1H), 6.93 (br s, 2H), 6.37 (d, J=5.46 Hz, 1H), 5.69(d, J=5.46 Hz, 1H), 4.77 (d, J=11.7 Hz, 1H), 4.52 (m, 3H), 4.39 (dd,J=7.8, 1.6 Hz, 1H), 4.17 (dd J=7.8, 3.9 Hz, 1H), 2.08 (s, 3H), 2.06 (s,3H), 2.05 (s, 3H), 2.03 (s, 3H); [M+H]⁺ 514.8; Elemental Analysis forC₁₉H₂₂N₄O₁₁S: calc'd: C, 44.36; H, 4.31; N, 10.89. found: C, 44.16; H,4.37; N, 10.69.

Example 50 Preparation of5-Amino-3-(3′-(R)-Butoxy-β-D-xylofuranosyl)-3H,6H-thiazolo[4,5-d]pyrimidin-2-one(146)

The required sugar acetic acid1,2,5-tri-O-acetyl-3-butoxy-D-xylofuranose α and β mixture (101) wasprepared as reported in Example 33.

Step 1: Preparation of5-Amino-3-(3′-(R)-Butox-2′,5′-di-O-acetyl-β-D-xylofuranosyl)-3H,6H-thiazolo[4,5-d]pyrimidin-2-one(145)

In a manner similar to Example 42, Step 1, the title compound 145 wasgenerated from acetic acid 1,2,5-tri-O-acetyl-3-butoxy-D-xylofuranose αand β mixture (101) and carried on crude to Step 2.

Step 2: Preparation of5-Amino-3-(3′-(R)-Butoxy-β-D-xylofuranosyl)-3H,6H-thiazolo[4,5-d]pyrimidin-2-one(146)

In a manner similar to Example 42, Step 2, 5.3 mg of the title compound146 was generated in 16% yield as a white solid: ¹H NMR (400 MHz,d₆-DMSO) δ 11.22 (br s, 1H), 6.92 (br s, 2H), 5.64 (d, J=5.46 Hz, 1H),5.60 (d, J=7.8 Hz, 1H), 5.08 (m, 1H), 4.39 (t, J=6.24 Hz, 1H), 4.07 (m,1H), 3.92 (t, J=7.02 Hz, 1H), 3.57 (m, 3H), 3.42 (m, 1H), 1.48 (m, 2H),1.33 (m, 2H), 0.88 (t, J=7.02 Hz, 3H); [M+H]⁺ 372.9; Elemental Analysisfor C₁₄H₂₀N₄O₆S.1.0H₂O.0.4MeOH: calc'd: C, 42.89; H, 5.90; N, 13.89.found: C, 43.18; H, 5.68; N, 13.65.

Example 51 Preparation of(3′S)-5-Amino-3-(3′-deoxy-3′-hydroxymethyl-β-D-ribofuranosyl)-3H,6H-thiazolo[4,5-d]pyrimidin-2,7-dione(148)

Step 1: Preparation of(3′S)-5-Amino-3-(3′-acetoxymethyl-2′,5′-di-O-acetyl-3′-deoxy-β-D-ribofuranosyl)-3H,6H-thiazolo[4,5-d]pyrimidin-2,7-dione(147)

In a manner similar to Example 42, Step 1, 224 mg of the title compound147 was generated from(3′S)-3-O-Acetoxymethyl-1,2,5-tri-O-acetyl-3-deoxy-α,β-D-ribofuranose[prepared according to the method of Cooperwood et al. Nucleosides,Nucleotides, and Nucleic Acids 2000, 19, 219-236 in which the enantiomerof the same compound was made] in 49% yield as an off-white solid: ¹HNMR (400 MHz, DMSO-d₆) δ 11.27 (s, 1H), 7.00 (s, 2H), 5.77 (m, 1H), 4.35(dd, J=11.7, 2.3, 1H), 4.26 (m, 1H), 4.15 (m, 2H), 4.07 (m, 3H), 2.013(s, 9H); [M+H]⁺ 457.3.

Step 2: Preparation of(3′S)-5-Amino-3-(3′-deoxy-3′-hydroxymethyl-β-D-ribofuranosyl)-3H,6H-thiazolo[4,5-d]pyrimidin-2,7-dione(148)

In a manner similar to Example 42, Step 2, 34 mg of the title compound148 was generated in 58% yield as an off-white solid: ¹H NMR (400 MHz,D₂O) δ 5.99 (m, 1H), 5.13 (m, 1H), 4.17 (m, 1H), 3.90 (m, 2H), 3.76 (m,2H), 2.93 (m, 1H); [M+H]⁺ 331.2; Elemental Analysis for C₁₁H₁₄N₄O₆S:calc'd: C, 35.20; H, 5.10; N, 14.93; S, 8.54. found: C, 35.17; H, 4.35;N, 14.73; S, 8.46.

Example 52 Preparation of5-Amino-3-(5′-deoxy-5′-hydroxymethyl-β-D-ribofuranosyl)-3H,6H-thiazolo[4,5-d]pyrimidin-2,7-dione(150)

Step 1: Preparation of5-Amino-3-(5′-O-acetoxymethyl-2′,3′-di-O-acetyl-5′-deoxy-β-D-ribofuranosyl)-3H,6H-thiazolo[4,5-d]pyrimidin-2,7-dione(149)

In a manner similar to Example 42, Step 1, 96 mg of the title compound149 was generated from5-O-acetoxymethyl-1,2,3-tri-O-acetyl-5-deoxy-α,β-D-ribofuranose[prepared according to the method of Pakulski et al. Polish J. Chem.1995, 69, 912-917] in 34% yield as a white solid: ¹H NMR (400 MHz,CDCl₃) δ 11.92 (s, 1H), 6.15 (d, J=6.4, 1H), 5.94 (s, 2H), 5.71 (m, 1H),4.91 (m, 1H), 4.40 (m 1H), 4.16 (m, 2H), 2.09 (s, 9H), 2.00 (m, 2H);[M+H]⁺ 457.0; Elemental Analysis for (C₁₇H₂₀N₄O₉S.0.25H₂O): calc'd: C,44.30; H, 4.48; N, 12.16; S, 6.96. found: C, 44.79; H, 4.62; N, 11.55;S, 6.59.

Step 2: Preparation of5-Amino-3-(5′-deoxy-5′-hydroxymethyl-β-D-ribofuranosyl)-3H,6H-thiazolo[4,5-d]pyrimidin-2,7-dione(150)

In a manner similar to Example 42, Step 2, 28 mg of the title compound150 was generated in 56% yield as a white solid: ¹H NMR (400 MHz,d₆-DMSO) δ 11.23 (s, 1H), 6.94 (s, 2H), 5.74 (m, 1H), 5.22 (m, 1H), 4.87(m, 1H), 4.40 (m, 1H), 4.00 (m, 2H), 3.44 (s, 3H), 1.72 (m, 2H); [M+H]⁺330.9.

Example 53 Preparation of5-Amino-3-[3′-deoxy-3′-O-p-toluenesulfonyl-β-D-xylofuranosyl]-3H,6H-thiazolo-[4,5-d]pyrimidine-2,7-dione(151)

Step 1: Preparation of5-Amino-3-[2′5′-di-O-acetyl-3′-deoxy-3′-O-p-toluenesulfonyl-β-D-xylofuranosyl]-3H,6H-thiazolo-[4,5-d]pyrimidine-2,7-dione(151)

In a manner similar to Example 42, Step 1, 24.6 mg of the title compound151 was generated in 12% yield as an off-white solid: ¹H NMR (400 MHz,CDCl₃) δ 11.89 (s, 1H), 7.84 (d, J=8.4, 2H), 7.40 (d, J=8.4, 2H), 6.22(d, J=4.4, 1H), 5.92 (br s, 2H), 5.75 (d, J=4.8, 1H), 4.95 (d, J=4.8,1H), 4.30 (m, 1H), 4.25 (d, J=6, 2H), 2.48 (s, 3H), 2.05 (s, 6H); [M+H]⁺555.3.

Example 54 Preparation of(3′R)-5-Amino-3-(3′-deoxy-3′-fluoro-β-D-xylofuranosyl)-3H,6H-thiazolo[4,5-d]pyrimidin-2,7-dione(153)

Step 1: Preparation of(3′R)-5-Amino-3-(2′-O-acetyl-5′-O-benzoyl-3′-deoxy-3′-fluoro-β-D-xylofuranosyl)-3H,6H-thiazolo[4,5-d]pyrimidin-2,7-dione(152)

In a manner similar to Example 42, Step 1, 149 mg of the title compound152 was generated from1,2-Di-O-acetyl-5-O-benzoyl-3-deoxy-3-(R)-fluoro-α,β-D-xylofuranose[prepared according to the method of Gosselin et al. CarbohydrateResearch 1993, 249, 1-17] in 24% yield as a yellow solid: ¹H NMR (400MHz, CDCl₃) δ 11.57 (s, 1H), 8.04 (d, J=6.8, 2H), 7.56 (t, J=7.6, 1H),7.43 (t, J=7.6, 2H), 6.35 (dd, J=22.4, 4.8, 1H), 5.92 (s, 2H), 5.32 (dd,J=51.6, 4.8, 1H), 5.20 (s, 1H), 4.79 (dd, J=11.2, 4, 1H), 4.59 (m, 2H),4.5 (m, 1H), 2.06 (s, 3H); [M+H]⁺ 465.3.

Step 2:(3′R)-5-amino-3-(3′-deoxy-3′-fluoro-β-D-xylofuranosyl)-3H,6H-thiazolo[4,5-d]pyrimidin-2,7-dione(153)

In a manner similar to Example 42, Step 2, 14.3 mg of the title compound153 was generated in 45% yield as white solid: ¹H NMR (400 MHz, DMSO-d₆)δ 11.41 (s, 1H), 6.97 (s, 2H), 5.77 (m, 1H), 5.19 (m, 1H), 4.98 (m, 1H),4.01 (m, 1H), 3.60 (m, 2H), 2.09 (s, 2H); [M+H]⁺ 318.9; Elementalanalysis for (C₁₀H₁₁FN₄O₅S.0.4EA.2H₂O): calc'd: C, 35.76; H, 4.71; N,14.3. found: C, 35.71; H, 3.68; N, 14.15.

Example 55 Preparation of3-Allyl-5-amino-3H,6H-thiazolo[4,5-d]pyrimidine-2,7-dione (154)

Step 1: Preparation of3-Allyl-5-amino-3H,6H-thiazolo[4,5-d]pyrimidine-2,7-dione (154)

In a manner similar to Example 15, Step 1, scheme1, 178 mg of the titlecompound 154 was generated in 35% yield as a pale yellow solid: ¹H NMR(400 MHz, DMSO-d₆) δ 10.26 (s, 1H), 6.09 (s, 2H), 5.06-5.01 (m, 1H),4.32 (dd, J=10.3, 1.5, 1H), 4.196 (dd, J=16.9, 1.5, 1H), 3.55 (d, J=4.4,2H); [M+H]⁺ 225.1.

Example 56 Preparation of5-Amino-3-pyridin-3-ylmethyl-3H,6H-thiazolo[4,5-d]pyrimidine-2,7-dione(155)

Step 1: Preparation of5-Amino-3-pyridin-3-ylmethyl-3H,6H-thiazolo[4,5-d]pyrimidine-2,7-dione(155)

In a manner similar to Example 15, Step 1, 143 mg of the title compound155 was generated in 24% yield as a white solid: ¹H NMR (400 MHz,DMSO-d₆) δ 10.33 (s, 1H), 7.76 (d, J=2.2, 1H), 7.68 (dd, J=2.2, 1.5,1H), 6.88 (m, 1H), 6.55 (s, 2H), 6.15 (s, 2H), 4.17 (s, 2H); [M+H]⁺276.1.

Example 57 Preparation of5-Amino-3-(4-chloro-but-2-enyl)-3H,6H-thiazolo[4,5-d]pyrimidine-2,7-dione(156)

Step 1: Preparation of5-Amino-3-(4-chloro-but-2-enyl)-3H,6H-thiazolo[4,5-d]pyrimidine-2,7-dione(156)

In a manner similar to Example 15, Step 1, 440 mg of the title compound156 was generated in a 63% yield as a light yellow solid: ¹H NMR (400MHz, DMSO-d₆) δ 10.33 (s, 1H), 6.90 (s, 2H), 5.82-5.74 (m, 1H),5.65-5.59 (m, 1H), 4.45 (d, J=7.0, 2H), 4.39 (d, J=7.8, 2H); [M+H]⁺273.1.

Example 58 Preparation of5-Amino-3-hexyl-3H,6H-thiazolo[4,5-d]pyrimidine-2,7-dione (157)

Step 1: Preparation of5-Amino-3-hexyl-3H,6H-thiazolo[4,5-d]pyrimidine-2,7-dione (157)

In a manner similar to Example 15, Step 1, 154 mg of the title compound157 was generated in a 35% yield as an off-white solid: ¹H NMR (400 MHz,DMSO-d₆) δ 11.03 (s, 1H), 6.88 (s, 2H), 3.74 (t, J=6.8, 2H), 1.61 (m,4H), 1.26 (m, 4H), 0.85 (t, J=6.8, 3H); [M+H]⁺ 269.31.

Example 59 Preparation of(1′R,2′S,3′R,4′R)-5-Amino-3-(2′,3′-dioxy-4′-hydroxymethyl-cyclopentan-1′-yl)-3H,6H-thiazolo[4,5-d]pyrimidine-2,7-dione(158)(1′R,2′S,3′R,4′R)—N′-[7-chloro-2-oxo-3-(2′,3′-O-isopropylidene-4′-vinyl-cyclopentan-1′-yl)-2,3-dihydro-thiozolo[4,5-d]pyrimidin-5-yl]-N,N-dimethyl-formamidine(85) (85 mg, 0.20 mmol) was dissolved in CH₃OH (3.0 mL) and H₂O (1.5 mL)at ambient temperature. The solution was cooled to 0° C. Sodiumperiodate (90 mg, 0.42 mmol) and Osmium tetroxide (2 mg, catalytic) werethen added to the solution. The reaction was stirred at 0° C. for 1 hand room temperature for 2 h before it was filtered and concentrated.The resulting mixture was dissolved in CH₂Cl₂ (10 mL), then washed withH₂O (2×10 mL). The organic layer was dried over MgSO₄, filtered, thenconcentrated.

The above product was dissolved in CH₃OH (3 mL) at ambient temperature.Sodium borohydride (12 mg, 0.32 mmol) was then added to the solution.The reaction was stirred for 1 h, then concentrated. The resultingmixture was dissolved in CH₂Cl₂, and washed with H₂O (2×10 mL). Theorganic layer was dried over MgSO₄, filtered, then concentrated.

The above product was dissolved CH₃OH (1 mL) and 2 M HCl (5 mL) atambient temperature. The reaction was stirred at reflux for 5 h, thenconcentrated. The resulting mixture was purified by reverse phase HPLCaffording 9.1 mg (13%) of 158 as a white solid: ¹H (400 MHz, CD₃OD) δ4.95 (m, 1H), 4.69 (dd, J=7.2, 5.6, 1H), 4.03 (t, J=5.2, 1H), 3.71 (dd,J=11.2, 6.4, 1H), 3.60 (dd, J=11.2, 6.4, 1H), 3.35 (s, 1H), 1.93-2.14(m, 3H).

Example 605-Amino-3-(2′-O-acetyl-3′-deoxy-β-D-ribofuranosyl)-3H,6H-thiazolo[4,5-d]pyrimidine-2,7-dione(160)

Preparation of5-Amino-3-(2′-O-acetyl-3′-deoxy-β-D-ribofuranosyl)-3H,6H-thiazolo[4,5-d]pyrimidine-2,7-dione(160)

To a suspension of nucleoside diacetate 121 (200.0 mg, 0.52 mmol) inacetone (5.0 mL), pH 7 phosphate buffer (2.5 mL), and H₂O (22.5 mL) wasadded Candida Arctica immobilized acrylic resin (0.10 g). The mixturedecolorized within 5 min after enzyme addition. The reaction was stirredat room temperature for 16 h. Celite (1.0 g) was added and, afterstirring for 10 min, the mixture was filtered through a pad of celite.The filter cake was rinsed with acetone (3×10 mL), and the acetone wasreduced in vacuo. The remaining aqueous layer was then heavily saltedwith solid NaCl. Ethyl acetate was added the biphasic mixture wasvigorously stirred for 30 min before separating. The organic layer wasdried over Na₂SO₄, decanted, concentrated, and purified via flashchromatography (SiO₂, 50-100% EtOAc-hexanes+2% MeOH). This afforded155.0 mg of monoacetate 160 (97%): ¹H (400 MHz, DMSO-d₆) δ 11.20 (s,1H), 6.94 (br s, 2H), 5.79 (d, J=2.4, 1H), 5.63 (d, J=8.0, 1H), 4.76 (t,J=6.0, 1H), 4.11 (dt, J=5.2, 10.4, 1H), 3.43-3.52 (m, 2H), 2.44-2.5 (m,1H), 2.05 (s, 3H), 1.95 (dd, J=6.0, 13.6, 1H); [M+H]⁺ m/z 342. Analysiscal'd for: C₁₂H₁₄N₄O₆S.0.5H₂O: C, 42.10; H, 4.12; N, 16.37; S, 9.37.Found: C, 42.30; H, 4.26; N, 16.37; S, 9.23.

Example 615-Amino-3-(2′,3′-dideoxy-β-D-ribofuranosyl)-3H,6H-thiazolo[4,5-d]pyrimidine-2,7-dione(165)

Step 1) Preparation of5-N-Acetylamino-3-(5′-tert-butyldimethylsily-2′-deoxy-2′-O-thiocarbonylimidazole-β-D-ribofuranosyl)-3H,6H-thiazolo[4,5-d]pyrimidine-2,7-dione(161)

To a mixture of alcohols 127 and 128 (247 mg, 0.541 mmol) in MeCN (8 mL)at rt was added TCDI (193 mg, 1.08 mmol). The reaction mixture wasstirred 18 h whereupon it was concentrated and submitted to flashchromatography (SiO₂, EtOAc), affording 150 mg (49%) of a mixture ofthiocarbamates 161 and 162 as a solid material: ¹H NMR (400 MHz,d₆-DMSO) δ 12.18 (br s, 1H), 11.72 (br s, 1H), 8.54 (s, 1H), 7.86 (s,1H), 7.10 (s, 1H), 6.36 (m, 1H), 6.04 (s, 1H), 4.38-4.43 (m, 1H),3.68-3.80 (m, 2H), 2.63-2.69 (br m, 1H), 2.36-2.41 (m, 1H), 2.17 (s,3H), 0.84 (s, 9H), 0.00 (s, 6H); [M+H]⁺ m/z 567.

Step 2) Preparation of5-N-Acetylamino-3-(5′-tert-butyldimethylsily-2′,3′-dideoxy-β-D-ribofuranosyl)-3H,6H-thiazolo[4,5-d]pyrimidine-2,7-dione(163)

To a mixture of thiocarbamates 161 and 162 (57 mg, 0.10 mmol) and Bu₃SnH(187 uL, 0.705 mmol) in PhMe (10 mL) at rt was added AIBN (1.6 mg, 0.010mmol). The reaction mixture was immersed into a 130° C. oil bath,stirred 20 min, concentrated and then submitted to flash chromatography(SiO₂, 60-80% EtOAc-CHCl₃), affording 28 mg (64%) of compound 163 as awhite solid: ¹H NMR (400 MHz, d₆-DMSO) δ 12.13 (br s, 1H), 11.80 (br s,1H), 6.11 (dd, J=8.4, 3.7, 1H), 3.95-4.02 (m, 1H), 3.64 (d, J=5.1, 2H),2.54-2.59 (m, 1H), 2.23-2.33 (m, 1H), 2.19 (s, 3H), 2.04-2.13 (m, 1H),1.94-1.97 (m, 1H), 0.82 (s, 9H), −0.01(6H); [M+H]⁺ m/z 441.

Step 3) Preparation of5-N-Acetylamino-3-(2′,3′-dideoxy-β-D-ribofuranosyl)-3H,6H-thiazolo[4,5-d]pyrimidine-2,7-dione(164)

A solution of siloxane 163 (84 mg, 0.19 mmol) in 2 M HF-MeCN (20 mL) wasstirred for 10 min, then concentrated and submitted to flashchromatography (SiO₂, 5-10% MeOH—CHCl₃) to afford 10 mg (16%) of alcohol164 as a white solid: ¹H NMR (400 MHz, d₆-DMSO) δ 12.15 (br s, 1H),11.78 (br s, 1H), 6.10 (dd, J=8.4, 4.0, 1H), 4.66 (t, J=5.9, 1H),3.91-3.97 (m, 1H), 3.46 (t, J=5.9, 2H), 2.51-2.58 (m, 1H), 2.21-2.32 (m,1H), 2.19 (s, 3H), 2.01-2.18 (m, 1H), 1.89-1.97 (m, 1H); [M+H]⁺ m/z 327.

Step 4) Preparation of5-Amino-3-(2′,3′-dideoxy-β-D-ribofuranosyl)-3H,6H-thiazolo[4,5-d]pyrimidine-2,7-dione(165)

To a solution of acetamide 164 (19 mg, 0.058 mmol) in MeOH (3 mL) at rtwas added K₂CO₃ (64 mg, 0.46 mmol). The reaction mixture was stirred 18h whereupon it was quenched with HOAc (53 uL), concentrated andtriturated with MeOH—H₂O to afford 6 mg (36%) of the title compound 165as a white solid: ¹H NMR (400 MHz, d₆-DMSO) δ 11.15 (s, 1H), 6.85 (br s,2H), 6.07 (dd, J=8.4, 4.4, 1H), 4.63 (t, J=5.9, 1H), 3.89-3.95 (m, 1H),3.46 (t, J=5.5, 2H), 2.44-2.48 (m, 1H), 2.17-2.27 (m, 1H), 2.01-2.11 (m,1H), 1.86-1.93 (m, 1H); [M+H]⁺ m/z 285.

Anti-Viral Activity of Compounds

A number of assays may be employed in accordance with the presentinvention in order to determine the degree of anti-viral activity of acompound of the invention such as cell culture, animal models, andadministration to human subjects. The assays described herein may beused to assay viral growth over time to determine the growthcharacteristics of a virus in the presence of a compound of theinvention.

In another embodiment, a virus and a compound of the invention areadministered to animal subjects susceptible to infection with the virus.The incidence, severity, length, virus load, mortality rate ofinfection, etc. can be compared to the incidence, severity, length,virus load, mortality rate of infection, etc. observed when subjects areadministered the virus alone (in the absence of a compound of theinvention). Anti-virus activity of the compound of the invention isdemonstrated by a decrease in incidence, severity, length, virus load,mortality rate of infection, etc. in the presence of the compound of theinvention. In a specific embodiment, the virus and the compound of theinvention are administered to the animal subject at the same time. Inanother specific embodiment, the virus is administered to the animalsubject before the compound of the invention. In another specificembodiment, the compound of the invention is administered to the animalsubject before the virus.

In another embodiment, the growth rate of the virus can be tested bysampling biological fluids/clinical samples (e.g., nasal aspirate,throat swab, sputum, broncho-alveolar lavage, urine, saliva, blood, orserum) from human or animal subjects at multiple time pointspost-infection either in the presence or absence of a compound of theinvention and measuring levels of virus. In specific embodiments, thegrowth rate of a virus is assayed by assessing the presence of virus ina sample after growth in cell culture, growth on a permissible growthmedium, or growth in subject using any method well-known in the art, forexample, but not limited to, immunoassay (e.g., ELISA; for discussionregarding ELISAs see, e.g., Ausubel et al., eds, 1994, Current Protocolsin Molecular Biology, Vol. I, John Wiley & Sons, Inc., New York at11.2.1), immunofluorescent staining, or immunoblot analysis using anantibody which immunospecifically recognizes the virus to be assayed ordetection of a virus-specific nucleic acid (e.g., by Southern blot orRT-PCR analysis, etc.).

In a specific embodiment, viral titers can be determined by obtainingbiological fluids/clinical samples from infected cells or an infectedsubject, preparing a serial dilution of the sample and infecting amonolayer of cells that are susceptible to infection with the virus(e.g. primary cells, transformed cell lines, patient tissue samples,etc) at a dilution of the virus that allows for the emergence of singleplaques. The plaques can then be counted and the viral titer expressedas plaque forming units per milliliter of sample.

In one specific embodiment, the growth rate of a virus in a subject canbe estimated by the titer of antibodies against the virus in thesubject. Antibody serum titer can be determined by any method well-knownin the art, for example, but not limited to, the amount of antibody orantibody fragment in serum samples can be quantitated by, e.g., ELISA.Additionally, in vivo activity of a Formula I compound can be determinedby directly administering the compound to a test animal, collectingbiological fluids (e.g., nasal aspirate, throat swab, sputum,broncho-alveolar lavage, urine, saliva, blood, or serum) and testing thefluid for anti-virus activity.

In embodiments where samples to be assayed for virus levels arebiological fluids/clinical samples (e.g., nasal aspirate, throat swab,sputum, broncho-alveolar lavage, urine, saliva, blood, or serum), thesamples may or may not contain in tact cells. Samples from subjectscontaining intact cells can be directly processed, whereas isolateswithout intact cells may or may not be first cultured on a permissivecell line (e.g. primary cells, transformed cell lines, patient tissuesamples, etc) or growth medium (e.g., LB broth/agar, YT broth/agar,blood agar, etc.). Cell suspensions can be cleared by centrifugation at,e.g., 300×g for 5 minutes at room temperature, followed by a PBS, pH 7.4(Ca⁺⁺ and Mg⁺⁺ free) wash under the same conditions. Cell pellets can beresuspended in a small volume of PBS for analysis. Primary clinicalisolates containing intact cells can be mixed with PBS and centrifugedat 300×g for 5 minutes at room temperature. Mucus is removed from theinterface with a sterile pipette tip and cell pellets can be washed oncemore with PBS under the same conditions. Pellets can then be resuspendedin a small volume of PBS for analysis.

In another embodiment, a compound of the invention is administered to ahuman subject infected with a virus. The incidence, severity, length,viral load, mortality rate of infection, etc. can be compared to theincidence, severity, length, viral load, mortality rate of infection,etc. observed in human subjects infected with a virus in the absence ofa compound of the invention or in the presence of a placebo. Anti-viralactivity of the compound of the invention is demonstrated by a decreasein incidence, severity, length, viral load, mortality rate of infection,etc. in the presence of the compound of the invention. Any method knownin the art can be used to determine anti-viral activity in a subjectsuch as those described previously.

Additionally, in vivo activity of a Formula I prodrug can be determinedby directly administering the compound to an animal or human subject,collecting biological fluids/clinical samples (e.g., nasal aspirate,throat swab, sputum, broncho-alveolar lavage, urine, saliva, blood, orserum) and testing the biological fluids/clinical samples for anti-viralactivity (e.g., by addition to cells in culture in the presence of thevirus).

Metabolism of Formula I Prodrugs

The Formula I prodrugs of the present invention must be metabolized toFormula II compounds and other compounds of the invention in the body ifthey are to serve as effective prodrugs. Hepatocyes often are used toassess the degree to which a compound may be transformed in the body ofan animal, and it is known that such transformations may vary withhepatocytes from different species in a way that reflects metabolism inthe whole animal. See Seddon T. et al., Biochem Pharmacol., 38(10),1657-65 (1989).

A study was undertaken to evaluate the metabolic stability of Formula Icompounds 14, 15, 13, 114, 30, 103, 67, 65, and 76 in the presence offresh cynomolgus monkey hepatocytes and monitor the formation of 6-oxymetabolites, i.e., Formula II compounds and other compounds of theinvention. For comparison, the metabolic stability of famciclovir wasalso assessed.

Preparation of Fresh Hepatocyte Suspension

Fresh cynomolgus monkey hepatocyte suspension (Lot #: Cy141) waspurchased from CellzDirect (Tucson, Ariz.). Hepatocyte Incubation Medium(serum-free, sterile) was purchased from In Vitro Technologies(Baltimore, Md.).

The cynomolgus monkey hepatocyte suspension was prepared from freshcynomolgus monkey hepatocytes in hepatocyte incubation medium at theconcentration of 1.25 million cells/mL. The final incubationconcentration (after test article addition) was 1.0 million cells/mL.

Preparation of Stock Solutions

Existing 100 mM stock solutions in DMSO were used. The concentrations ofthe test articles were checked using UV-vis microplate reader.Correction coefficients were determined using the absorbance of afreshly prepared DMSO stock of 122.

Incubations

Reaction suspensions were prepared in removable 96-well tubes, eachcontaining 320 μL of fresh cynomolgus monkey hepatocyte suspension atthe density of 1.25 million cells per mL and 40 μL of hepatocyteincubation medium. The above mixtures were pre-incubated open at 37° C.,95% humidity and 5% CO₂ for 30 minutes. Reactions were initiated by theaddition of 40 μL of test article at 10× concentration to each tube toachieve the final concentrations of 50 μM for the test article(s) and 1million/mL cell density. The reaction suspension in each tube was mixedby inverting the tube several times. Aliquots of 50 μL from eachreaction suspension were distributed into six additional removable96-well tubes (one tube per time point taken at 15, 30, 45, 60, 90, and120 minutes). The open tubes were incubated at 37° C. under 95% humidityand 5% CO₂.

Preparation of Samples for Analysis

At predetermined time points, reactions were terminated by the additionof 150 μL of the stop solution to each tube containing 50 μL of thereaction suspension. The composition of the stop solution was thefollowing: 15 mL of acetonitrile (containing 1 μg/mL nebularine as aninternal standard and 0.1% formic acid) combined with 1 mL water.

The calibration curves were prepared in the following way. To 80 μL ofcell suspension (at the cell density of 1.25 million/mL) 10 μL ofhepatocyte incubation medium and 10 μL of the appropriate concentrationof the compound in hepatocyte incubation medium were added. Immediatelyfollowing the compound addition, 300 μL of the stop solution (see above)was added.

All quenched samples were kept on wet ice until they were processed foranalysis. Then they were mixed using a bench top Multi-Tube Vortexer(VWR Scientific Products) for approximately 30 seconds, and centrifugedat 4,000 rpm (3,220 rcf) for 10 minutes at 4° C. Clear supernatant (100μL) was transferred into a clean deep well 96-well plate, evaporated todryness under nitrogen, reconstituted in 100 μL of 90:10water:acetonitrile, and analyzed for the parent form and metabolites ofthe test article using an appropriate LC/MS/MS method.

Bioanalysis

The compounds were quantified on an API3000 LC/MS/MS instrument in theESI-Positive MRM (multiple reaction monitoring) mode. The summary of theresults on Formula I prodrug degradation and product generation is givenin Table 1.

TABLE 1 Concentration of the Metabolized Product Formed in CynomolgusMonkey Hepatocytes after 2 hrs Incubation of 50 μM of a Formula IProdrug Formula I Metabolized Compound Product Product Concentration(μM) Response 14 122 9.7 + 15 122 31.8 ++++ 13 122 24.2 +++ 114 134 21.7+++ 30 117 21.5 +++ 103 141 13.1 ++ 57 157 1.7 + 65 148 14.7 ++ 76 13221.4 +++ Famciclovir Penciclovir 5.9 +

In fresh cynomolgus monkey hepatocytes compounds 14, 15, 13, 114, 30,103, 67, 65, and 76 as well as famciclovir are metabolized to yield thecorresponding 6-oxy metabolites: 122 from the first three Formula Iprodrugs and 134, 117, 141, 157, 148, and 132, respectively. Famciclovirproduces penciclovir.

IFN-α Induction from Peripheral Blood Mononuclear Cells (PBMC)

Peripheral blood mononuclear cells (PBMCs) are prepared by standardmethods from human blood and are primarily comprised of monocytes, NKcells, circulating dendritic cells and both T and B cells. Briefly, theyare purified by density gradient centrifugation from a buffy coat, whichis the component of whole blood that contains leukocytes and platelets.In turn, buffy coats are prepared by centrifuging whole blood andisolating the thin cream colored layer between the upper plasma layerand the lower red blood cell portion of the separated mixture.

PBMC Purification

Freshly collected donor buffy coats were obtained from the San DiegoBlood Bank. PBMCs were isolated from the buffy coats usinghistopaque-1077 gradient (Sigma), essentially as described in themanufacturer's protocol. Buffy coats were transferred into 50 mlcentrifuge tubes and PBS added to a total volume of 35 ml. Next 10 mlhistopaque-1077 was underlayed at the bottom of each tube, which werethen centrifuged at 259×g for 30 minutes at room temperature withoutbrake in a 5804 R centrifuge (Eppendorf). The top PBS level from eachtube was removed and discarded and the buffy coat layer transferred to afresh tube. The total volume was made up to 50 ml with PBS and the tubeswere then centrifuged for 10 minutes at 259×g at room temperature. Thecells were washed an additional 3 times with PBS in this manner.

The cell (PBMC) pellet was then resuspended in 30-40 ml complete (RPMI1640) media. PBMCs were seeded at either 2.5 or 7.5×10⁶ cells/mlcomplete media (1× and 3× seedings, respectively) and allowed to restovernight before compound exposure for 24 hours. The cells and mediawere then collected, centrifuged for 5 minutes at 735×g in a 5415 Cmicrofuge (Eppendorf) at room temperature and the supernatant analyzedby IFN-α ELISA. The ability of Formula I prodrugs, Formula II compounds,and other compounds of the invention to demonstrate favorable oraldelivery characteristics and to induce immune responses whenadministered by a selected route can be compared with the results ofsimilar experiments with compounds described in the literature. Herebyincorporated by reference in their entireties are U.S. Pat. Nos.5,041,426 and 4,880,784, and U.S. patent application Ser. No. 10/861,430(U.S. Patent Application Publication No. US 2005/0070556), whichdisclose, inter alia, IFN-α induction of isatoribine.

Accordingly, relative activities of the compounds of the invention areexpressed as a percentage of the level of IFN-α induction by either 32or 100 μM isatoribine.

ELISA Protocol

Human IFN-α ELISA (#KHC4012) was performed as described in Biosourceprotocol. However, to ensure that readings were within the linear rangeof detection, PBMC supernatant samples were typically diluted 1:3(2.5×10⁶ cells/ml seeding) or 1:15 (7.5×10⁶ cells/ml seeding) and thenanalyzed together with undiluted supernatant samples. The concentrationin each sample was calculated from the O.D. by reference to a standardcurve.

The compounds of the invention exhibited IFN-α induction from PBMCsrelative to isatoribine in the following ranges:

0-10%: Compounds 142, 157, 141, 148, and 33

11-50%: Compounds 152 and 165

51-100%: Compounds 32, 160, 130, and 137

>100%: Compounds 133, 34, 147, 121, 122, 134, 117, 132, and 153.

Comparison to Isatoribine

The results demonstrate the remarkable superiority of 134 and 122 toisatoribine with respect to enhancement of IFN-α production from humanPBMCs in vitro. FIG. 1 and FIG. 2 show plots of pg/ml IFN-α induced inhuman PBMCs from compounds 134 and 122 vs pg/ml IFN-α induced by anidentical concentration of isatoribine. The results may be summarized asfollows.

At 1×PBMC seeding, both 134 and 122 induce significantly andsubstantially more IFN-α production at 100 μM test compound than doesisatoribine, especially for weak responders to isatoribine (left panelon FIGS. 1 and 2). When seeding is increased to 3×, the differencebetween the amount of IFN-α produced at 100 μM test agent is lessclear-cut although visually apparent and statistically significant(right panel, FIGS. 1 and 2). When the concentration of both 134 and 122is 32 μM at 3× seeding, however, both 134 and 122 dramatically andunexpectedly outperform isatoribine (right panel and inserts of FIGS. 1and 2).

It is to be understood that the foregoing description is exemplary andexplanatory in nature, and is intended to illustrate the invention andits preferred embodiments. Through routine experimentation, the artisanwill recognize apparent modifications and variations that may be madewithout departing from the spirit of the invention. Thus, the inventionis intended to be defined not by the above description, but by thefollowing claims and their equivalents.

1-40. (canceled)
 41. A method of treating a tumor or cancer in a patientin need thereof comprising administering to said patient atherapeutically effective amount of a compound selected from the groupconsisting of:

or a pharmaceutically acceptable salt or tautomer thereof.
 42. Themethod according to claim 41, wherein the tumor or cancer is caused by avirus.
 43. The method according to claim 41, wherein the tumor or canceris selected from the group consisting of carcinomas, sarcomas, andleukemias.
 44. The method according to claim 43, wherein the carcinomais a mammary, colon, bladder, lung, prostate, stomach, or pancreascarcinoma.
 45. The method according to claim 43, wherein the leukemia isa lymphoblastic or myeloid leukemia.
 46. The method according to claim41, further comprising administering to said patient a therapeuticallyeffective amount of a therapeutic agent selected from the groupconsisting of antibiotics, antiemetic agents, antidepressants,antifungal agents, anti-inflammatory agents, antiviral agents,anticancer agents, immunomodulatory agents, β-interferons, alkylatingagents, hormones, and cytokines.
 47. A method of treating a tumor orcancer in a patient in need thereof comprising administering to saidpatient a therapeutically effective amount of a compound that is

or a pharmaceutically acceptable salt thereof.
 48. The method accordingto claim 47, wherein the tumor or cancer is caused by a virus.
 49. Themethod according to claim 47, wherein the tumor or cancer is selectedfrom the group consisting of carcinomas, sarcomas, and leukemias. 50.The method according to claim 49, wherein the carcinoma is a mammary,colon, bladder, lung, prostate, stomach, or pancreas carcinoma.
 51. Themethod according to claim 49, wherein the leukemia is a lymphoblastic ormyeloid leukemia.
 52. The method according to claim 47, furthercomprising administering to said patient a therapeutically effectiveamount of a therapeutic agent selected from the group consisting ofantibiotics, antiemetic agents, antidepressants, antifungal agents,anti-inflammatory agents, antiviral agents, anticancer agents,immunomodulatory agents, β-interferons, alkylating agents, hormones, andcytokines.
 53. The method according to claim 47, wherein the compound isorally administered to said patient.
 54. A method of treating a tumor orcancer in a patient in need thereof comprising administering to saidpatient a therapeutically effective amount of a compound that is

or a pharmaceutically acceptable salt or tautomer thereof.
 55. Themethod according to claim 54, wherein the tumor or cancer is caused by avirus.
 56. The method according to claim 54, wherein the tumor or canceris selected from the group consisting of carcinomas, sarcomas, andleukemias.
 57. The method according to claim 56, wherein the carcinomais a mammary, colon, bladder, lung, prostate, stomach, or pancreascarcinoma.
 58. The method according to claim 56, wherein the leukemia isa lymphoblastic or myeloid leukemia.
 59. The method according to claim54, further comprising administering to said patient a therapeuticallyeffective amount of a therapeutic agent selected from the groupconsisting of antibiotics, antiemetic agents, antidepressants,antifungal agents, anti-inflammatory agents, antiviral agents,anticancer agents, immunomodulatory agents, β-interferons, alkylatingagents, hormones, and cytokines
 60. The method according to claim 54,wherein the compound is administered to said patient in an oral orparenteral dosage form.